Pluripotency determining factors and uses thereof

ABSTRACT

Pluripotency determining factors are described which act intracellularly and maintain a pluripotent cell in a pluripotent state in the absence of gp130 activation, which maintain or confer pluripotency of a human stem cell, which maintain or confer pluripotency of a mouse ES cell, and which maintain or confer pluripotency of a stem cell from a non-permissive strain of mice. The factors and vectors encoding or activating the factors are used to maintain and derive pluripotent cells, especially of higher mammals, including humans.

The present invention relates to pluripotency determining factors andtheir uses. In particular the invention relates to maintaining andderiving cultures of pluripotent cells of mouse, human and otherspecies.

From certain strains of mouse, embryonic stem (ES) cells may be derivedand maintained in a pluripotent state in culture by provision of acytokine that activates signal transduction through theLIF-receptor/gp130 complex. This approach is, however, limited to miceand is not sufficient for propagation of human pluripotent cells.

The discovery of the extracellular activity ESRF (Dani et al, 1998) hasprovided evidence for a LIF-receptor/gp130-independent pathway formaintaining ES cell identity. However, ESRF is incompletelycharacterised and the molecular nature of extracellular regulators otherthan gp130 cytokines is unknown.

In addition, the transcriptional determination of pluripotency is notfully understood. Important roles have been assigned to the gp130 targetSTAT3 and the POU factor Oct-4, but neither of these alone is sufficientto specify ES cell identity.

WO 02/097090 describes the identification of ECAT (ES cell associatedtranscript) genes by interrogating nucleotide sequence databases, andsubsequent analysis of the expression pattern of murine and human ECATgenes. WO 02/097090 also describes the use of probes to identify EScells and selective isolation of ES cells using ECAT genes. Oct-4 is anECAT gene and a specific marker of pluripotent cells, though isexpressed also in oocytes, cleavage embryos and egg cylinder stages.

WO 01/66697 describes culture of ES cells in medium free of animal serumand in the presence of fibroblast growth factor. Preferably the ES cellsare grown in the presence of a fibroblast feeder layer.

WO 97/30151, in the name of the present applicants, describes ESRF, acytokine capable of inhibiting differentiation of ES cells.

WO 96/22693 relates to the maintenance of self-renewing haematopoieticstem cells. In particular, WO 96/22693 describes an “F factor” capableof maintaining haematopoietic stem cells in an undifferentiated stateand supporting proliferation of undifferentiated haematopoietic stemcells.

Berger C. N. and Sturm K. S., Growth Factors 1997, Volume 14, pages145-159, describes self-renewal of ES cells in the absence of exogenousLIF and feeder cells. Berger and Sturm do not, however, describe theisolation of any factor able to promote self-renewal of ES cells underthese conditions.

Genbank Accession Numbers AK010332, 2001, and AK022643, 2000, providesequences of mouse and human cDNAs for homeobox domain containingproteins. No function is ascribed to these molecules.

It is known that pluripotency of human embryonic stem cells andembryonic germ cells is sustained by co-culture with a feeder layer ofheterologous cells (Amit et al, Dev. Biol. 2000) or extracts therefrom(Xu et al. Nat. Biotech 2001). However, dependency on feeder cells givesrise to a number of problems. Co-culture of feeder cells and pluripotentcells may mask compromised culture conditions. Co-culture riskscontamination of the pluripotent culture/differentiated product derivedtherefrom. To produce feeder cell extract, which is an alternative tousing the cells, a separate feeder cell population must be maintainedand processed, and the extract stored. As the extract isuncharacterised, it represents another source of contamination of theproduct. Analysis and manipulation of differentiation of pluripotentcells is compromised by the presence of feeder cells or undefinedextracts.

In relation to many other species, in particular porcine, bovine andrat, it is still not possible to derive and stably maintain pluripotentES cells in culture.

An object of the present invention is to provide improved culture ofmouse and human pluripotent cells, and an object of specific embodimentsof the invention is to remove the dependency on feeder cells inpluripotent cell culture. A further object is to provide for derivationand maintenance of pluripotent cells from other species.

Accordingly, the present invention provides mammalian pluripotencydetermining factors, exemplified in a first embodiment by a factor thatmaintains pluripotency in a mammalian cell.

In a culture of ES cells, the factor has been found to be sufficient toconfer pluripotency of those cells. According to the invention, thefactor may be maintained in a pluripotent cell at a level that resultsin maintenance of a self-renewing pluripotent phenotype. The factor maybe endogenous—a cellular level of the factor being maintained byactivation of an endogenous gene—or introduced into the cell so as toenable stable maintenance of the pluripotent phenotype of the cells.

A particular factor of the invention acts intracellularly, maintains acell in a pluripotent state and, in cell culture, is constitutivelyactive. In this context, reference to intracellular action indicatesthat the site of activity lies within the cell. The factor is thus not,for example, a ligand for a cell surface receptor, as is the case withLIF and other cytokines, and it can act independently of signalling fromgp130 receptors.

Further pluripotency determining factors of the invention maintainpluripotent cells, cultured in ES cell medium lacking gp130 activators,in a pluripotent state. Thus, the factor is able to maintain a mammaliancell in a pluripotent state in the absence of gp130 activation and canpromote maintenance of pluripotency in culture medium lacking cytokinesthat activate gp130-receptor complexes. In examples below we havedemonstrated that cells expressing preferred factors of the inventionare liberated from the requirement for gp130 stimulation and showreduced or absent differentiation in response to retinoic acid,3-methoxybenzamide and aggregation—hence their differentiation issuppressed in response to these stimuli. The factor of specific examplesis, further, not a downstream transcriptional target of gp130. Thefactors of the invention have been isolated and are suitable formaintenance of animal pluripotent cells, animal cells including those ofmouse, human, primate, sheep, rat, pig, cow and other animals. Onefactor of the invention maintains pluripotency of a human ES, EC or EGcell or other human pluripotent cell. Another factor of the inventionmaintains pluripotency of a mouse ES, EC or EG cell and actsintracellularly and is constitutively active. A further factor of theinvention allows the continuous propagation in culture of pluripotentcells from non-permissive strains of mice.

An advantage of the invention lies in the ability to stably proliferatepluripotent cells in culture. Factors of the invention can be used incultures to maintain human and/or mouse cells in a proliferative,pluripotent state. The invention further offers the possibility toisolate and maintain ES cells from species other than human andmouse—not possible hitherto.

A factor of a specific embodiment is a transcription factor. It actsinside the nucleus, resulting in self-renewal of the ES cell. Apreferred factor, described and used in an example below, contains ahomeodomain

In use of one factor of the invention, the factor maintains cells in apluripotent state in the absence of a feeder layer or a feeder cellextract and in the absence of a gp130 cytokine. Thus, supply of thefactor is sufficient to maintain pluripotency of cells in culture. Thefactor can be added to standard ES cell culture medium or introduceddirectly into cells, for example by electroporation or microinjection,or produced in the cell by a variety of methods disclosed herein. Inspecific examples of the invention, described in more detail below,vectors that express the factor support cytokine independent ES cellpropagation both via episomal transfection of cells and upon chromosomalintegration into cells; a further option, not specifically exemplified,is for expression of an endogenous sequence encoding the factor to beinduced or increased so as to enable ES cell self renewal.

In a further embodiment of the invention, the factor is identifiable byits property of maintaining LIF non-responsive cells in a pluripotentstate.

The factor suitably is a polypeptide having from 200 to 400 amino acids.Specifically, a mouse pluripotency determining factor of the inventionis represented by SEQ ID NO 2, a human pluripotency determining factorby SEQ ID NO 4, a rat pluripotency determining factor by SEQ ID NO 6,and a macaque pluripotency determining factor by SEQ ID NO 8.

A second aspect of the invention lies in a factor which maintains a cellin a pluripotent state, acts intracellularly and comprises ahomeodomain, in particular a homeodomain that has at least 50% sequenceidentity with the homeodomain from SEQ ID NO: 2, 4, 6 or 8 or, inrelation to a factor for cells of a given species, one that has at least50% sequence identity with the homeodomain of pluripotency determiningfactor of the same species. Generally, a homeodomain is around 60 aminoacids in length and the factor of the invention comprises a homeodomainin which any 20 amino acid fragment has at least 35% sequence identitywith the homeodomain of SEQ ID NO: 2, 4, 6 or 8. Preferably, the factormaintains LIF non-responsive cells in a pluripotent state.

A factor of a further embodiment is one which maintains culturedepiblast cells from non-permissive strains of mice in a pluripotentstate in the absence of feeder cells and in the absence of feeder cellextract. A second such factor is one that maintains human embryo-derivedcells (including ES and EG cells) in a pluripotent state in the absenceof feeder cells and in the absence of feeder cell extract.

The avoidance of feeder cells or feeder cell extracts has the benefit ofreducing contamination of the culture by unwanted cells. Mouse cellshave been used as feeder cells for human pluripotent cell cultures, andso the invention significantly reduces and may eliminate the risk ofcontamination of the human pluripotent cell population by non-humancells. If ES cell derived products are to be used in transplantationtherapies, a guarantee of absence of non-human cells is likely to beessential, both for initial isolation of cells and also for subsequentpropagation.

Also provided by the invention are conjugates of the factor with anotherfunctional domain. A first such conjugate comprises first and seconddomains, wherein the first domain comprises a factor of the inventionand the second domain promotes uptake of the first domain into a celle.g. protein transduction domains of antennapedia, HIV-1 TAT protein orHSV-1 VP22 (Schwarze et al., 2000). Hence, the conjugate can be used asa culture medium component. It is further optional for the second domainto include a nuclear localization sequence to assist in trafficking thefactor to the nucleus after uptake into a cell.

To enable release of the first domain from the second in use, the firstdomain of a preferred conjugate may be cleaved from the second domaininside the cell. The first domain may be linked to the second domain bya di-sulphide bridge—which allows release of the first domain in thereducing environment of the cell. The first domain may be linked to thesecond domain covalently, allowing the link to be cleaved by a proteasepresent in the cell. It is further optional for the second domain tocomprise sequence permitting regulation of the activity of the factor,for example a steroid hormone receptor (Picard, 2000).

Further isolated polypeptides of the invention include (a) polypeptidemolecules comprising an amino acid sequence as set out in SEQ ID NO: 2,4, 6 or 8; (b) naturally occurring variants of (a); (c) orthologues of(a) or (b), and (d) biologically active and diagnostically ortherapeutically useful fragments, analogues and derivatives thereof.

The polypeptide of the present invention may be a recombinantpolypeptide, a natural polypeptide or a synthetic polypeptide,preferably a recombinant polypeptide.

The terms “fragment”, “derivative” and “analogue” when referring to thepolypeptide of the invention mean a polypeptide which retainsessentially the same biological function or activity as suchpolypeptide. The fragment, derivative or analogue of the polypeptide ofthe invention may be (i) one in which one or more of the amino acidresidues are substituted with a conserved or non-conserved amino acidresidue (preferably a conserved amino acid residue) and such substitutedamino acid residue may or may not be one encoded by the genetic code, or(ii) one in which one or more of the amino acid residues includes asubstituent group, or (iii) one in which the mature polypeptide is fusedwith another compound, such as a compound to increase the half-life ofthe polypeptide (for example, polyethylene glycol), or (iv) one in whichthe additional amino acids are fused to the mature polypeptide, forexample to facilitate purification of the mature polypeptide. Suchfragments, derivatives and analogues are deemed to be within the scopeof those skilled in the art from the teachings herein.

The polypeptides of the invention can also be used to prepare antibodiesthat specifically bind to pluripotency determining factor epitopes,peptides or polypeptides. Methods for preparing polyclonal andmonoclonal antibodies are well known in the art (see, for example,Harlow and Lane, Using Antibodies: A Laboratory Manual, Cold SpringHarbor, N.Y., 1999; and Hurrell, J. G. R., Ed., Monoclonal HybridomaAntibodies: Techniques and Applications, CRC Press, Inc., Boca Raton,Fla., 1982, which are incorporated herein by reference). Polyclonalantibodies can be generated from a variety of animals, such as horses,cows, goats, sheep, dogs, chickens, rabbits, mice, and rats.

The immunogenicity of a pluripotency determining factor polypeptide maybe increased through the use of an adjuvant, such as alum (aluminiumhydroxide) or Freund's complete or incomplete adjuvant. Polypeptidesuseful for immunization also include fusion polypeptides, such asfusions of pluripotency determining factor or a portion thereof with animmunoglobulin polypeptide or with maltose binding protein. Thepolypeptide immunogen may be a full-length molecule or a portionthereof. If the polypeptide portion is “hapten-like”, such portion maybe advantageously joined or linked to a macromolecular carrier (such askeyhole limpet hemocyanin (KLH), bovine serum albumin (BSA) or tetanustoxoid) for immunization.

As used herein, the term “antibodies” includes polyclonal antibodies,affinity-purified polyclonal antibodies, monoclonal antibodies, andantigen-binding fragments, such as F(ab′)₂ and Fab proteolyticfragments. Genetically engineered intact antibodies or fragments, suchas chimeric antibodies, Fv fragments, single chain antibodies and thelike, as well as synthetic antigen-binding peptides and polypeptides,are also included. Non-human antibodies may be humanized by graftingonly non-human CDRs onto human framework and constant regions, or byincorporating the entire non-human variable domains (optionally“cloaking” them with a human-like surface by replacement of exposedresidues, wherein the result is a “veneered” antibody). In someinstances, humanized antibodies may retain non-human residues within thehuman variable region framework domains to enhance proper bindingcharacteristics. Through humanizing antibodies, biological half-life maybe increased, and the potential for adverse immune reactions uponadministration to humans is reduced. Alternative techniques forgenerating or selecting antibodies useful herein include in vitroexposure of lymphocytes to pluripotency determining factor protein orpeptide, and selection of antibody display libraries in phage or similarvectors (for instance, through use of immobilized or labelledpluripotency determining factor protein or peptide).

Antibodies are defined to be specifically binding if they bind to apluripotency determining factor polypeptide with a binding affinity(K_(a)) of 10⁶ M⁻¹ or greater, preferably 10⁷ M⁻¹ or greater, morepreferably 10⁸ M⁻¹ or greater, and most preferably 10⁹ M⁻¹ or greater.The binding affinity of an antibody can be readily determined by knowntechniques (for example, by Scatchard analysis).

A variety of assays known to those skilled in the art can be utilized todetect antibodies which specifically bind to pluripotency determiningfactor proteins or peptides. Exemplary assays are described in detail inUsing Antibodies: A Laboratory Manual, Harlow and Lane (Eds.), ColdSpring Harbor Laboratory Press, 1999. Representative examples of suchassays include: concurrent immunoelectrophoresis, radioimmunoas say,radioimmuno-precipitation, enzyme-linked immunosorbent assay (ELISA),dot blot or Western blot assay, inhibition or competition assay, andsandwich assay. In addition, antibodies can be screened for binding towild-type versus mutant pluripotency determining factor protein orpeptide.

The polypeptides of the present invention additionally include thepolypeptides of SEQ ID NO: 2, 4, 6 and 8 (in particular the maturepolypeptide) as well as polypeptides which have at least 50% similarity(preferably at least 50% identity) to the polypeptide of SEQ ID NO: 2,4, 6 or 8 and more preferably at least 90% similarity (more preferablyat least 90% identity) to the polypeptide of SEQ ID NO: 2, 4, 6 or 8 andstill more preferably at least 95% similarity (still more preferably atleast 95% identity) to the polypeptide of SEQ ID NO: 2, 4, 6 or 8 andalso include portions of such polypeptides with such portion of thepolypeptide generally containing at least 30 amino acids and morepreferably at least 50 amino acids. “Similarity” between twopolypeptides is determined by comparing the amino acid sequence and itsconserved amino acid substitutes of one polypeptide to the sequence of asecond polypeptide. Various different approaches are known for thecalculation of sequence similarity and identity. Generally, a suitableway to perform these calculations is to run database searches using aprogram such as Smith-Waterman, BLAST or FASTA, and use one orpreferably two or even three similarity tables. The Blosum and PAM(Point Accepted Mutation) matrices are suitable amino acids similaritymatrices for database searching and sequence alignment. IfSmith-Waterman or FASTA is used then it is relevant to ensure the opengap penalty is large enough, and if the initial runs do not uncover anyhomologous sequences it can be appropriate to try a differentalgorithm—this is particularly true if you started with one of theheuristic algorithms, BLAST or FASTA.

A yet further aspect of the invention lies in compositions containingthe factor or the conjugate, and include a pharmaceutical compositioncomprising the factor or conjugate as described together with apharmaceutically acceptable carrier; and a cell culture mediumcomprising the factor or conjugate as described.

Cell culture medium, containing a factor and/or a conjugate of theinvention may additionally contain one or more components selected fromthe groups consisting of GMEM, serum, serum replacement, essential aminoacids, .beta.-mercaptoethanol, pyruvate and glutamine.

The culture medium optionally further contains an activator of gp130,and suitable activators include cytokines that act at the LIF receptor,e.g. LIF, and IL-6 in combination with soluble IL-6 receptor. Theculture medium can be used for maintenance of pluripotent ES cells,especially ES, EC and EG cells and for self-renewal of pluripotentcells, especially ES, EC and EG cells. The medium can further be usedfor maintenance of somatic cells, especially somatic stem cells, and ina particular embodiment of the invention there is provided a method ofself-renewal of somatic cells comprising culturing the cells in thepresence of the medium and preferably propagating the cells in themedium. A still further use of the culture medium is for maintenanceand/or propagation of cells derived from pluripotent cells.

Another application of the invention lies in expansion of cellpopulations, for example in expansion of somatic cells. An ex vivotherapy that can be carried out exploiting the invention comprisesremoving a population of cells from a patient, culturing the cells inculture medium of the invention so as to expand the cell population andthereafter transplanting or reintroducing the cells into the patient.

Further culture methods of the invention comprise maintaining and/orpropagating cells using a combination of a factor of the invention andexpression of Oct-4 and/or activation of Oct-4. The factor can beintroduced as the factor per se or via a conjugate or using vectors asherein described. Oct-4 expression can be achieved by upregulatingexpression of an endogenous Oct-4 gene or by introducing an expressionvector comprising Oct-4 into the cells. A combination of the factor ofthe invention and Oct-4 can be used to maintain and/or self-renew and/orpropagate cells. In particular this combination can be used to provideenhanced pluripotent cell self-renewal and/or enhanced maintenance ofpluripotency. The combination may further be used for derivation ofpluripotent cell populations. In a specific embodiment of the inventionit has been found that a combination of the factor and Oct-4 leads toimproved pluripotent cell self-renewal, and this improvement isexploited in the derivation of new pluripotent cell lines, especially ofhuman cells but also of other animals, including murine such as rat andmouse; primate such as monkey, especially macaque; porcine; sheep; andbovine pluripotent cells,

Pluripotent cells, especially ES cells, are suitably derived bytransfecting blastocyst cells prior to plating or at plating, forexample at the time that feeder cells are conventionally used, or at thetime that the inner cell mass is spread onto a plate. The transfectioncan be carried out with cells on feeder layers or not on feeder layers,though there is an advantage in avoiding any contact between the cellsto be transfected and feeder cells. It is known that transgenesis ofpluripotent cells is achievable at this stage, including forpre-implantation embryos, and hence transfection so that a factor of theinvention is expressed is used to derive stable, pluripotent cellpopulations.

Another use of the technology described lies in cellular and nuclearreprogramming, and accordingly the invention also provides a method ofreprogramming the nucleus of a somatic cell, comprising contacting thecell with a factor of the invention and activating gp130 signalling inthe cell, or comprising contacting the cell with a factor of theinvention and expressing Oct-4 in the cell. For example, thereprogramming is carried out by transfecting the cell with a firstvector that contains a nucleotide sequence encoding a factor of theinvention, and with a second vector that contains a nucleotide sequenceencoding a LIF receptor ligand or contains a nucleotide sequenceencoding Oct-4.

The invention additionally provides polynucleotides encoding the factorsof the invention. In use of the factor, there may be a number ofdifferent methods proposed for control or direction of expression of thefactor so as to promote pluripotency of cells in culture or elsewhere.It is thus optional to include in the polynucleotide a sequence whichregulates expression of the factor. This sequence may be a promoter, andcan also be a promoter whose activity is regulated. Furtherpolynucleotides of the invention encode the conjugates described above.

To control the level of factor in a given cell, a gene encoding thefactor and present in the cell may be activated. Both endogenous andheterologous promoters and other regulatory elements may be used, aswell as factors that act on these regulatory elements. One approach isto use a polynucleotide for inserting a regulatory sequence into thegenome of a cell, wherein the regulatory sequence when insertedregulates expression of a gene encoding the factor. In this way, anendogenous gene can be turned on or its expression maintained. Theregulatory sequence can be inserted by homologous recombination into theresident gene encoding the factor. The regulatory sequence may be aregulated promoter—for example an inducible promoter can be used so thatthe factor is expressed when culture medium contains an inducer;removing the inducer results in reduced expression of the factor andloss of pluripotent phenotype. Sequences which enable removal orinactivation of the promoter may also be included, to allow furthercontrol of expression of the factor.

Another method of controlling the level of a factor in a cell is todesign or select sequence specific DNA binding polypeptide domains whichrecognize sequences in the promoter of the endogenous PDF gene, forexample by using phage display (Isalan and Choo, 2001 Methods inEnzymology 340 p 593-609). Such sequence specific DNA binding domainsmay be used to activate or maintain transcription of the endogenous PDFgene by fusion to transcriptional activation domains. Alternatively,fusion of such sequence specific DNA binding polypeptide domains tosilencer domains may be used to reduce PDF expression. Additionally,these fusions may be linked to other domains that promote uptake of thefusion protein into the cell. In this way molecules which act on theendogenous PDF gene itself may be administered to cells or expressed incells.

The polynucleotides of the present invention may be in the form of RNAor in the form of DNA, which DNA includes cDNA, genomic DNA, andsynthetic DNA. The DNA may be double-stranded or single-stranded, and ifsingle stranded may be the coding strand or non-coding (anti-sense)strand. The coding sequence which encodes the mature polypeptide may beidentical to the coding sequence shown in SEQ ID NO: 1, 3, 5 or 7 may bea different coding sequence which coding sequence, as a result of theredundancy or degeneracy of the genetic code, encodes the same maturepolypeptide as the DNA.

The isolated polynucleotides of the invention may encode (a) polypeptidemolecules comprising an amino acid sequence as set out in SEQ ID NO: 2,4, 6 or 8; (b) naturally occurring variants of (a); (c) orthologues of(a) or (b), and (d) biologically active and diagnostically ortherapeutically useful fragments, analogues and derivatives thereof.

The polynucleotide which encodes for these polypeptides may also includeadditional coding sequence and/or non-coding sequence such as introns ornon-coding sequence 5′ and/or 3′ of the coding sequence for the maturepolypeptide. Thus, references to “polynucleotide” include reference to apolynucleotide which includes only coding sequence for the polypeptideas well as a polynucleotide which includes additional coding and/ornon-coding sequence.

The present invention further relates to variants of the herein abovedescribed polynucleotides which encode fragments, analogues andderivatives of the polypeptide having the specified amino acid sequence.The variants of the polynucleotide may be naturally occurring variantsof the polynucleotide or non-naturally occurring variants of thepolynucleotide.

Thus, the present invention includes polynucleotides as shown in SEQ IDNO: 1, 3, 5 or 7 encoding the same mature polypeptide as well asvariants of such polynucleotides which variants encode for a fragment,derivative or analogue of the factor. Such nucleotide variants includedeletion variants, substitution variants and addition or insertionvariants.

As herein above indicated, the polynucleotide may have a coding sequencewhich is a naturally occurring variant of the coding sequence shown inSEQ ID NO 1, 3, 5 or 7. As known in the art, an alternate form of apolynucleotide sequence may have a substitution, deletion or addition ofone or more nucleotides, which does not substantially alter the functionof the encoded polypeptide.

The present invention further relates to polynucleotides which hybridiseto one or more of the herein above-described sequences if there is atleast 70%, preferably at least 90%, and more preferably at least 95%identity between the sequences. The present invention particularlyrelates to polynucleotides which hybridise under stringent conditions tothe herein above-described polynucleotides. As herein used, the term“stringent conditions” means hybridisation will occur only if there isat least 95% and preferably at least 97% identity between the sequences.The polynucleotides which hybridise to the herein above describedpolynucleotides in a preferred embodiment encode polypeptides whicheither retain substantially the same biological function or activity asthe mature polypeptide encoded by the cDNAs of SEQ ID NO: 1, 3, 5 or 7.

Alternatively, the polynucleotide may have at least 20 bases, preferablyat least 30 bases, and more preferably at least 50 bases which hybridiseto a polynucleotide of the present invention and which has an identitythereto, as herein above described, and which may or may not retainactivity. For example, such polynucleotides may be employed as probesfor the polynucleotide of SEQ ID NO: 1, 3, 5 or 7, for example, forrecovery of the polynucleotide or as a diagnostic probe or as a PCRprimer.

Still further provided by the present invention are vectors for use inexpressing the factor of the invention. A first vector comprises apolynucleotide of the invention as described above. A vector describedand used in an example of the invention, set out in more detail below,is designed for transfection of a cell such that the factor is expressedin the cell and the transfected cell may be maintained in a pluripotentstate. Generally, such vectors comprise the following operatively linkedelements: a transcription promoter; a DNA segment comprising apolynucleotide of the invention; and a polyadenylation signal. A furtheroption is for the vector additionally to encode for a selectable marker,with selection used to identify cells that have taken up and areexpressing the vector. As an example, the vector can encode antibioticresistance, with addition of antibiotic to the culture used to identifysuccessful transfectants. Following transfection of the cell, the vectormay be chromosomally integrated or maintained extra-chromosomally andthe coding sequence on the vector expressed so that a level of thefactor is maintained in the cell sufficient to keep the cell in apluripotent state. A particular vector is designed for transfection of acell expressing or containing polyoma large T antigen. Such a vector mayhave a polyoma origin of replication, such that the vector is stablymaintained in the cell when polyoma large T antigen is present.

Further control and flexibility may be conferred on the system by usinga promoter in the vector whose activity can be controlled. Thus,following transfection, the promoter may be in a non-active state,allowing the user to choose when to activate the promoter, for exampleusing an inducible promoter the inducer can be added to culture medium.As an alternative, the promoter may be initially active but such thatits activity can be reduced or turned off by addition of a suppressorsubstance to culture medium—this enables the user to determine, after aperiod of maintaining cells in a pluripotent state, to halt expressionof the pluripotency determining factor from the vector, allowing thecells to differentiate and substantially clearing the culture of anyremaining pluripotent cells. In preparation of differentiated progenythere is a need to remove undifferentiated cells, and thus this elementof the invention provides this capability. A suitable system is the tetregulatory system (Baron & Bujard, 2000). The vector may additionally beconstructed such as to enable its subsequent removal, or removal of thesequence encoding the factor, by site specific recombination.

A vector of a further embodiment of the invention comprises a nucleotidesequence which encodes a regulatory factor, wherein the regulatoryfactor regulates a gene encoding the factor of the invention. Theregulatory factor can be used to activate or turn on or otherwiseincrease expression from the gene, which may be an endogenous gene ormay be located extra-chromosomally. This particular vector has a numberof uses. A transgenic line of animals may be prepared in which a geneencoding the pluripotency determining factor is substantially turnedoff. The transgenic animals can then be used to generate embryos andpluripotent cells explanted therefrom and transfected with this vectorsuch that the regulatory factor of the vector turns on expression of thepluripotency determining factor, allowing maintenance of a culture ofpluripotent cells. Alternatively, a binary transgenic animal can begenerated containing both the vector for the regulatory factor and alsothe regulatable gene for a pluripotency determining factor. The lattercan then be induced in vivo to mobilize stem cells.

A principal use of the factor, whether directly or by expression of anucleotide sequence encoding the factor, is in maintenance of cells in apluripotent state. The polynucleotide may be a sequence endogenous tothe cell, for example the native sequence may be activated, to achievethe maintenance of pluripotency, or a nucleotide sequence introducedinto the cell may be used. The introduced sequence may also be on aplasmid. A nucleotide encoding the factor may also be on a stablyintegrated transgene.

The invention hence provides a method of maintaining a pluripotent cellpopulation, comprising administering to that population a factor of theinvention. A further culture method is to maintain a cell in apluripotent state by activating a gene in the cell that encodes a factorof the invention. A yet further culture method for maintaining a cell ina pluripotent state comprises maintaining or increasing expression of agene in the cell that encodes a factor according to the invention.

The pluripotency determining factors of the invention can also be usedto increase the potency of a somatic or non-pluripotent cell. Hence, amethod of the invention comprises increasing the potency of a cell byexposing the cell to a pluripotency determining factor, this exposureoptionally being carried out by introducing the factor into the cell orexpressing in the cell a nucleotide sequence encoding the factor. Theincrease in potency of the cell may be such as to re-programme thenucleus of the cell, leading to the somatic or non-pluripotent cellbeing converted into a pluripotent stem cell. Alternatively, the effectof the factor of the invention upon the cell may be to increase itspotency but not to increase its potency so far as to render the cellpluripotent. Once a cell has had its potency increased, the cell maythen be subjected to differentiation down a different lineage, and hencethe factor of the invention can be useful in respecifying the lineage ofa given cell.

The factor enables a number of different strategies for obtainingpluripotent cells to be pursued. In one such strategy, a pluripotentnon-human cell is isolated by creating a transgenic animal, the animalcontaining a construct in which a nucleotide sequence encoding thefactor of the invention is under the control of a regulated promoter. Atransgenic embryo is then obtained from the transgenic animal and acell, such as an epiblast cell or a primordial germ cell, obtained fromthe embryo. By activation of the promoter the factor is expressed in thecell and, subsequently, pluripotent cells can be isolated. In thisapproach, therefore, the transgenic animal is made as an initial stepand this approach is particularly suitable where procedures exist forcreation of transgenic animals. In another approach, which can forexample be adopted in species in which it is not possible or in which itis technically more demanding to make transgenic animals, a nucleotideencoding the factor of the invention is introduced into a cell in afreshly isolated cell population and a pluripotent cell isolatedthereafter. In this latter approach, cells from the freshly isolatedcell population are transfected quickly, that is to say without allowingthe cells to remain in culture for sufficient time for them to havedifferentiated before at least one cell is transfected such that thefactor is expressed in that cell. As mentioned, this approach can haveparticular utility in deriving and maintaining pluripotent cells frommammals or other animals in which it is not possible to make transgenicanimals. Hence, also included within the invention are transgenicanimals obtainable by these methods and transgenic animals containing anucleotide sequence encoding a factor according to the invention undercontrol of a regulatable promoter.

Methods are described herein in which the factor and functionalanalogues, variants, fragments and derivatives are used to promotemaintenance of pluripotency. Another benefit of the invention and theidentification of pluripotency determining factors is that we now havethe opportunity to devise antagonists to these pluripotency determiningfactors, which antagonists can be used to inhibit the activity ofpluripotency determining factors or otherwise act as antagonists to suchfactors. Antagonists to the factors can be used to promotedifferentiation of a pluripotent cell, and this has utility for examplein situations in which it is desired to eliminate pluripotent cellseither from a culture or in vivo. The present invention also enablesproduction of animals that are dominant negative for the factor, such asanimals containing dominant negative variants or other dominant negativemutants of the gene encoding the factor.

Thus, also provided by the invention is a composition comprising apharmaceutically acceptable carrier plus an antagonist of a factor ofthe invention. Antagonists can be used to effectively remove pluripotentcells prior to introduction of pluripotent cell-derived progeny into apatient, such as in cell therapy, avoiding for example teratomas and/orother tumours due to contamination of transplanted material bypluripotent cells. This can be applied in vivo or in vitro. Another usefor the antagonist is as a contraceptive. A further use of theantagonist of the invention is generally for treatment of tumours, inparticular tumours associated with inappropriate expression of a factorof the invention.

Cells are provided by the invention in which the expression or activityof a factor of the invention has been manipulated. These cells can bemaintained in a pluripotent state. A first cell of the present inventionexpresses a factor of the invention for a period of time in excess of 2weeks. Further cells of the invention are (1) a cell comprising a vectorof the invention, (2) a pluripotent, human cell, maintained in apluripotent state in the absence of feeder cells and in the absence offeeder cell extract, and (3) a cell comprising a gene encoding a factoraccording to the invention wherein the gene has been activated.

Cells of the invention can also be used in a screen for molecules thatinterfere with the function of the factor of the invention. One suchscreen comprises culturing a pluripotent cell in the presence of afactor of the invention, the factor being present for example by beingprovided in cell culture medium or by being expressed in the cell,culturing the cell in the presence of a test substance and observingwhether there is any alteration in the pluripotent phenotype of thecell. This assay can be used to select for antagonists of the factor orfor other molecules that interfere with its activity. The screen canalso be used to identify test substances with functions of inducingdifferentiation of the cell.

Cells of the invention can additionally be used in assays for drugdiscovery. Cells of the invention may also be used for cell therapy, andthus a method of the invention comprises using the factor of theinvention to derive and/or maintain pluripotent cells, deriving cellsfor cell therapy therefrom and using those cells in cell therapy.

A screening method of the invention, for screening a cDNA library,comprises performing the screen in pluripotent cells, and selecting fora self-renewing phenotype. The screen can use pluripotent cells that arecytokine non-responsive, and has advantage in that both extrinsic andintrinsic factors can be thereby identified.

The invention further provides a method of screening a cDNA librarycomprising carrying out a complementation assay in pluripotent cells.The method can be employed to identify a cDNA which confers aself-renewing phenotype.

The invention yet further provides a method of screening for a factorthat maintains a pluripotent cell in a pluripotent state, comprising: —

-   -   providing a pluripotent cell which expresses a pluripotency        determining factor of the invention, which cell is stably        maintained in a pluripotent state and in which expression of the        factor can be controlled;    -   carrying out a manipulation of the cell;    -   reducing expression of the pluripotency determining factor; and    -   selecting for a cell that is maintained in a pluripotent state.

Manipulations can be designed so as to result in a screen for extrinsicfactors, cell surface receptors, cDNAs or other factors, and suitablemanipulations include a genetic manipulation of the cell and amanipulation of culture medium or culture conditions in which the cellis cultured. The screen can thereby be made suitable for identifying afactor which controls expression of an endogenous gene coding for apluripotency determining factor of the invention or for a further factorthat maintains the cell in a pluripotent state. Preferably, expressionof the factor is controlled by regulating a regulatable promoter inoperative combination with a nucleotide sequence encoding the factor,enabling expression of the factor to be reduced, so as to remove theactivity of the pluripotency determining factor, by adding a repressorto medium in which the cell is cultured. Cells that remain pluripotentcan then be isolated and characterized to determine the identity of thefactor that has substituted for pluripotency determining factor.

A further screen of the invention is one for identifying regulation ofthe endogenous gene, and the invention further provides a method ofscreening for a factor that regulates expression of an endogenous genewhich codes for a pluripotency determining factor of the invention,comprising: —

-   -   (1) analysing regions of the genome that flank the endogenous        gene for presence of transcription factor binding sites, and    -   (2) screening fragments of those regions with an extract from ES        cells to identify a component of the extract that bind to the        fragments.

Regions of the genome that flank the endogenous gene and directpluripotent cell restricted expression of the factor are preferablyidentified, suitably by transfection of reporter constructs, so that thescreen can identify one or more components in the ES cell extract thatare putative transcription factors for pluripotency determining factor.Further analysis of the results of the screen can include research intowhether control of the putative transcription factor, such as via anextracellular substance, has already been identified. The thusidentified controlling factors can be used to derive and/or maintainpluripotent mammalian cells.

The term “orthologue” denotes a polypeptide or protein obtained from onespecies that is the functional counterpart of a polypeptide or proteinfrom a different species. Sequence differences among orthologues are theresult of speciation.

The term “allelic variant” denotes any of two or more alternative formsof a gene occupying the same chromosomal locus. Allelic variation arisesnaturally through mutation, and may result in phenotypic polymorphismwithin populations. Gene mutations can be silent (no change in theencoded polypeptide) or may encode polypeptides having altered aminoacid sequence.

The term “expression vector” denotes a DNA molecule, linear or circular,that comprises a segment encoding a polypeptide of interest operablylinked to additional segments that provide for its transcription. Suchadditional segments may include promoter and terminator sequences, andmay optionally include one or more origins of replication, one or moreselectable markers, an enhancer, a polyadenylation signal, and the like.Expression vectors are generally derived from plasmid or viral DNA, ormay contain elements of both.

The term “isolated”, when applied to a polynucleotide molecule, denotesthat the polynucleotide has been removed from its natural geneticenvironment and is thus free of other extraneous or unwanted codingsequences, and is in a form suitable for use within geneticallyengineered protein production systems. Such isolated molecules are thosethat are separated from their natural environment and include cDNA andgenomic clones. Isolated DNA molecules of the present invention aregenerally free of other genes with which they are ordinarily associated,but may include naturally occurring 5′ and 3′ flanking regionscontaining regulatory elements such as promoters, enhancers andterminators. The identification of associated regions will be evident toone of ordinary skill in the art (see for example, Dynan and Tijan,Nature 316:774-78, 1985). When applied to a protein, the term “isolated”indicates that the protein is found in a condition other than its nativeenvironment, such as apart from blood and animal tissue. In a preferredform, the isolated protein is substantially free of other proteins,particularly other proteins of animal origin. It is preferred to providethe protein in a highly purified form, i.e., greater than 95% pure, morepreferably greater than 99% pure.

The term “operatively linked”, when referring to DNA segments, denotesthat the segments are arranged so that they function in combination fortheir intended purposes, e.g. transcripts initiate in the promoter andproceed through the coding segment to the polyadenylation site.

The term “polynucleotide” denotes a single- or double-stranded polymerof deoxyribonucleotide or ribonucleotide bases read from the 5′ to the3′ end. Polynucleotides include RNA and DNA, and may be isolated fromnatural sources, synthesized in vitro, or prepared from a combination ofnatural and synthetic molecules.

The term “complement” with respect to polynucleotide molecules denotespolynucleotide molecules having a complementary base sequence andreverse orientation as compared to a reference sequence. For example,the sequence 5′ ATGCACGGG 3′ is complementary to 5′ CCCGTGCAT 3′.

The term “degenerate nucleotide sequence” denotes a sequence ofnucleotides that includes one or more degenerate codons (as compared toa reference polynucleotide molecule that encodes a polypeptide).Degenerate codons contain different triplets of nucleotides, but encodethe same amino acid residue (i.e., GAU and GAC triplets each encodeAsp).

The term “promoter” denotes a portion of a gene containing DNA sequencesthat provide for the binding of RNA polymerase and initiation oftranscription. Promoter sequences are commonly, but not always, found inthe 5′ non-coding regions of genes.

The invention is now described in the following specific embodiments,illustrated by the accompanying drawings in which: —

FIG. 1 shows the plasmid pPyCAGIP.

FIG. 2 shows integration and subsequent removal of a loxP flankedpluripotency determining factor transgene.

FIG. 3 shows sequence analysis of PDF cDNA in which: —

(A) shows alignment of the PDF homeodomain with the most closely relatedrepresentatives of several different classes of homeodomain protein (SEQID NOS:10-23). The alignment was generated with Clustal W and shaded,using MacBoxshade, with respect to PDF: dark grey, identical in allsequences; mid grey, identical to PDF; light grey, similar to PDF.Percentage identities to the PDF homeodomain are shown on the right.

(B) shows pairwise comparison of mouse (Mm) PDF and the human (Hs)orthologue (hypothetical protein FLJ12581) (SEQ ID NOS:2 AND 4).Identical residues are boxed.

(C) shows that the 3′ UTR of the mouse PDF cDNA contains a B2 repeat(SEQ ID NOS:24 and 25). Numbering is according to PDF cDNA and B2sequence accession number K00132. The split RNA polymerase III promoterof B2 is boxed. Asterisks indicate bases that differ between the PDFcDNA described here and the RIKEN cDNA AK010332.

(D) demonstrates the activity of the human orthologue of PDF in mouse EScells. LRK1 cells were transfected with pPyCAGIP (no insert) or aderivative carrying a hPDF ORF insert. The number of alkalinephosphatase positive colonies with no surrounding differentiated cellswere counted and expressed as the percentage formed in the absence ofcytokine relative to the number formed in the presence of IL6/sIL6R.

FIG. 4 shows that expression in vitro is restricted to pluripotentcells, in which: —

(A) shows comparative hybridisation of RNA from MEFs and from MEF/EScell co-cultures used for library construction. 1 μG pA⁺ RNA was loadedper lane and hybridised with probes for PDF cDNA (left), GAPDH (right)and PDF ORF (middle). Positions of migration of RNA markers of theindicated sizes (kb) are shown to the left.

(B) shows PDF transcripts detected by in situ hybridisation of a MEF/EScell co-culture (left) and an undifferentiated colony of cellssurrounded by differentiated cells in an E14Tg2a culture (right); barsare 50 μm.

(C) shows expression in cell lines is restricted to ES, EC and EG cells.RNAs used were PSMB, PC13.5, F9, PSA4, P19, EC cells; CGR8, ES cells;PE, Parietal endoderm; PYS, parietal yolk sac; NIH3T3, fibroblasts;BAFB03, pro-B cells; MEL, erythroleukaemia; B9, plasmacytoma.

(D) shows PDF expression is repressed upon ES cell differentiation. RNAswere from E14Tg2a cells induced to differentiate by application of RA orMBA for the number of days shown.

(E) shows a lack of detectable expression of PDF in adult tissues. RNAsused were 1, epidydimis; 2, testes; 3, CGR8; 4, adipose; 5, kidney; 6,liver; 7, heart; 8, spleen; 9, brain; 10, bone marrow; 11, tongue; 12,eye; 13, oviduct; 14, thymus; 15, skeletal muscle; 16, skin; 17, ovary;18, seminiferous vesicle; 19, lung.

(F) shows human PDF RNA is expressed in EC cells. RNAs used were fromembryonal carcinoma (GCT27C4) and lymphoid (Jurkat) cells. Northern blotanalysis was performed by sequential hybridisation using probes for PDF,GAPDH and Oct4 (C, D), PDF and GAPDH (E) and hPDF and GAPDH (F).

FIG. 5 shows expression of PDF in vivo in which PDF mRNA was visualisedby in situ hybridisation using an ORF probe and: —

(A) shows pre-implantation embryos. The top series shows embryos of 1,2, 6 and 8 cells and a late morula. The bottom series shows blastocystsat early, expanded, hatched and implanting stages. All panels are shownat the same magnification.

(B) shows E11.5 genital ridges from female (top) and male (bottom)embryos.

FIG. 6 illustrates the reversibility of gp130 independent self-renewal,in which: —

(A) provides a diagram of construct. The CAG cassette directs expressionof a PDF-IRES-pacpA transcript. This is followed by a transcriptionterminator sequence (STOP; SPA C2 MAZ) and an egfppA cassette. The loxPsites are positioned in the second exon of the CAG cassette and betweenthe terminator sequence and egfp such that following Cre mediatedrecombination CAG directs expression of egfp.

(B) shows Northern blot analysis of transgene transcripts before(PDF-IRES-pac, pac probe) and after (gfp) Cre excision. The blot washybridised sequentially with the indicated probes. E, E14Tg2a; EF1,E14Tg2a subclone carrying the Floxed transgene; EF1C1, EF1 subclonefollowing Cre-mediated excision.

(C) shows that reversal of PDF expression restores LIF dependentself-renewal. ES cells (ZIN40), ES cells expressing the Floxed PDFtransgene and their Cre-excised derivative lines were analysed followingplating at clonal density in the indicated culture conditions; 0, noaddition; LIF, 100 u/ml LIF, hLIF-05, LIF antagonist sufficient to block10 units/ml LIF. After 6 days culture the percentage of alkalinephosphatase positive colonies lacking discernible differentiated cellswas quantitated.

(D) shows the morphology of PDF expressing cells and their Cre-excisedderivative in the absence of cytokine (0), in 100 u/ml LIF (LIF) or inthe presence of LIF antagonist sufficient to block 10 units/ml LIF(hLIF-05). Cells were plated at clonal density and examined after 4 daysculture.

(E) shows Northern blot analysis of RNA from cultures of E14Tg2aderivatives expressing the floxed transgene (EF4) or a Cre excisedsubclone (EF4C3) prepared at 0, 1, 2, 3 or 4 days following exposure toRA or MBA and hybridised to Oct4.

FIG. 7 shows the reversibility of gp130 independent ES cell identity, inwhich: —

(A) shows differentiation by aggregation. E14Tg2a derivatives expressingthe floxed transgene (EF4) or Cre excised subclone (EF4C3) were assessedfor cardiac differentiation by placing individual embryoid bodies in awell of a 48 well dish and scoring each well daily for the presence ofbeating cells.

(B) shows differentiation by aggregation in the presence of retinoicacid. E14Tg2a and derivatives expressing the floxed transgene (EF4) orCre excised subclone (EF4C3) were assessed for neurogenesis by TuJimmunohistochemistry.

(C) shows the contribution of Cre deleted cells to mid gestation embryo.EF1C1 cells were injected into an MF1 blastocyst and after transfer to afoster mother examined at E9.5 for green fluorescence.

FIG. 8 shows the relationship of PDF to other known mediators ofself-renewal, in which: —

(A) shows that the proportion of cytokine independent coloniescorrelates with the level of episomal PDF expression. LRK1 cells weretransfected with episomal vectors directing increasing levels ofexpression of PDF (pPyPPGK<pPyPCAG<pPyCAGIP). Following 12 days ofselection, the proportion of self-renewing colonies in the absence ofcytokine is expressed relative to the number in the presence ofIL6/sIL6R. Data are the average of two independent experiments.

(B) shows that PDF is not a STAT3 target. ES cells expressing chimaericGCSFR-gp130 variant molecules in which all four STAT3 binding sites wereabolished by mutation of tyrosine codons to phenylalanine (Y126-275F) orin which the negative regulatory tyrosine was similarly mutated (Y118F)were stimulated with LIF (L) or GCSF (G) for the indicated time (mins)before RNA preparation. Northern blot analysis was performed bysequential hybridisation with probes against PDF, GAPDH or the STAT3target gene SOCS3.

(C) shows that PDF cannot substitute for Oct4 in self-renewal. ES cellsin which doxycycline responsive Oct4 transgene sustains self-renewal(ZHBTc4.1) were transfected with linearised pPyCAGIP derivativescarrying no insert (MT), Oct4 (Oct) or PDF (PDF) and cultured in thepresence of LIF under conditions in which the transgene remainsexpressed (0) or is switched off (Dox). Following puromycin selection,plates were stained and the percentage of undifferentiated coloniesdetermined.

FIG. 9 illustrates the maintenance of pluripotent GCT 27X-1 hEC cells inconditioned medium in which: —

(A) shows the GCT 27X-1 pluripotent hEC cell line, which is routinelymaintained on a feeder layer of mitotically inactivated STO cells.

(B) depicts pluripotent cultures, shown at a routine density, and whichcan also be maintained in the absence of feeder cells but with theaddition of conditioned medium derived from the yolk sac carcinoma cellline hEC 44 to 25% v/v in the routine culture medium.

(C) shows that withdrawal of this conditioned medium from feeder-freeGCT 27X-1 cell cultures, shown at a clonal density, results in theinitiation of cell differentiation and loss of pluripotent phenotype.Photographed under phase-contrast optics, 10×(A, B), 4×(C).

FIG. 10 shows the Northern blot analysis of endogenous hPDF expressionin GCT 27X-1 hEC cells, in which poly A+ mRNA (3-3.5 μg) prepared fromGCT 27X-1 cells cultured on STO feeder cells (27X-1+STO), and withoutfeeder cells but with the addition of conditioned medium (27X-1-STO+CM),was separated on a denaturing gel and analysed by Northern blothybridisation with a cDNA probe (˜900 bp) specific to the candidatehuman PDF sequence. Poly A+ mRNAs prepared from a STO cell culture (STO)and from an E14Tg2a mouse ES cell culture (E14Tg2a) were included ascontrols for hybridisation. A major transcript of ˜2.4 kb and a lesserhybridising transcript of ˜5.6 kb were detected in both GCT 27X-1cultures, confirming endogenous expression of the hPDF gene inpluripotent hEC cell cultures. A very weak hybridisation band of ˜2.2 kbdetected in the mouse ES cell line is indicative of a homologoustranscript in mouse pluripotent cells. As expected no hybridisationsignal was detected for STO cell mRNA. Autoradiographic exposure isshown for 8 hrs at −70° C. with intensifying screens.

FIG. 11 provides a flow chart describing experimental strategy todemonstrate the maintenance of self-renewal in a human pluripotent cellline expressing the hPDF cDNA. (CM=conditioned medium)

FIG. 12 illustrates the floxed hPDF and GFP control vector constructs,in which: —

(A) shows the 8.8 kb Floxed hPDF construct, which contains the openreading frame for the hPDF cDNA (˜900 bp) operating under a humancytomegalovirus immediate early enhancer (CMV IE) and the human actinpromoter, upstream of a bicistronic reporter cassette providing forIRES-mediated expression of the puromycin resistance gene. The enhancedGFP (eGFP) reporter gene lies downstream of loxP sites, providing forvisualisation by fluorescence where Cre-mediated recombination occurs.

(B) shows the pPyCAGegfpIP (“GFP control”) vector, which is a similarconstruct in which the hPDF sequence in (A) is replaced by the eGFPsequence upstream of the IRES-puromycin reporter cassette and was usedto provide control “non hPDF”-expressing transfectants.

FIG. 13 illustrates the morphological evaluation of transfectant GCT27X-1 colonies. Stable transfectant Floxed hPDF and GFP control colonieswere established following 10-15 days in puromycin selection culturewithout STO feeder cells, in the presence and absence of conditionedmedium. Following Leishman's staining colony morphology was scored as(A) “Tight”: those colonies maintaining a tight pluripotent hECphenotype, (B) “Medium”: those colonies starting to differentiate whilemaintaining some hEC phenotype and (C) “Loose”: those colonies that werecompletely loose and differentiated. Photographed under phase-contrastoptics, 4×.

FIG. 14 illustrates the morphology of hPDF-expres sing hEC cells in aclonal assay, in which a-d show hPDF-expres sing hEC clonal linesexpanded continuously in the presence (hPDF D7) and absence (hPDF E7) ofconditioned medium from transfection, which were cultured at a clonaldensity for 12-14 days with (+CM) and without (−CM) conditioned medium;and e-h show a transfected GFP control clone (GFP C15) and wild type hECcells (27X-1), which were also cultured under the same conditions. Inthe absence of conditioned medium, only hPDF-expressing hEC cells areable to maintain a pluripotent hEC phenotype, while GFP control and wildtype hEC cells are induced to differentiate. Photographed underphase-contrast optics, 10×.

FIG. 15 shows the self-renewal of hPDF-expressing hEC cells in a clonalassay. hPDF-expressing hEC clonal lines (hPDF D7 and E7), a transfectedGFP control clone (GFP C15) and wild type hEC cells (27X-1), werecultured at a clonal density for 12-14 days in the presence (+CM, a-h)and absence (−CM, i-p) of conditioned medium. Pluripotent hEC cellswithin colonies were identified by indirect immunofluorescent stainingfor the CD30 marker (b, d, f, h, j, l, n, p), shown here withcomparative Hoechst UV fluorescence for all cells within the same colony(a, c, e, g, i, k, m, o). In the absence of conditioned medium, onlyhPDF-expressing hEC colonies are able to maintain a pluripotentphenotype, while control transfected and wild type hEC cells are inducedto differentiate unless maintained in conditioned medium. The hPDF E7colonies displayed a generally tighter morphology and stronger detectionof the CD30 marker when compared with hPDF D7 cells, in the absence ofconditioned medium. Colonies were visualised using 535/50 nm (CD30) andUV 330-380 nm (Hoechst) filters, at 20× magnification.

FIG. 16 illustrates the self-renewal of hPDF-expressing hEC cells inroutine culture. The hPDF-expressing hEC cell line E7 has been expandedin the continuous absence of both STO feeder cells and conditionedmedium since the time of transfection. This clonal cell line, shown herefollowing routine passaging, continues to display self-renewal of hECcells after more than 8 weeks of culture under these conditions.Photographed under phase-contrast optics, 10×.

EXAMPLE 1 Pluripotency Determining Factor Design of Screen

A functional screen of a cDNA library was designed to meet two criteria.Firstly, the frequency with which cDNA can be introduced into cells andmaintained for the duration of the screen was to be high enough so thatscreening a complex library of 10⁵-10⁶ independent cDNAs is practical.This is fulfilled by the combined use of LRK1 cells (described below)which express polyoma large T antigen and the polyoma origin containingvector pPyCAGIP (FIG. 1). Of the episomal vectors we have tested, thisplasmid gives the highest levels of cDNA expression (data not shown).Furthermore, compared to selections with Blasticidin S, Hygromycin B andZeocin, Puromycin selection allows the most rapid elimination ofuntransfected ES cells with 95% untransfected cells killed within 1 day(data not shown). Thus, a potential source of confounding biologicalactivities is quickly removed from the cultures. Secondly, the propertyfor which one is screening was to be present in the cell line beingtransfected at a sufficiently low level to enable the screen to proceedwithout a prohibitively high background. Ideally this property is absentfrom but expressible in the host cell line. LRK1 cells fulfil thiscritical criteria. During differentiation in vitro, expression from theLIF locus increases (Rathjen et al. 1990) and a number of ES coloniescan be seen to emerge from the differentiated cell monolayer (Dani etal. 1998). Much of these re-emergent colonies appear due to the actionof LIF (Dani et al. 1998). E14/T cells which retain responsiveness toLIF exhibit this background self-renewal following differentiation. Incontrast, LRK1 cells which have lost responsiveness to cytokines actingvia LIFR produce a far smaller number of colonies following thewithdrawal of IL6 and sIL6R. This reduction in background self-renewalis sufficient to allow a library screen to be performed using thesecells.

Preparation of LRK1 Cells

A cell line which could be supertransfected at high frequency withpolyoma origin containing vectors was required in order to enable cDNAlibrary screening to proceed at high efficiency. The cell line E14/T, aderivative of E14Tg2a cells which had been transfected with the plasmidpMGD20neo (Gassmann et al. 1995) and which supported replication of asupertransfecting plasmid was chosen as this line retained the abilityto differentiate efficiently upon withdrawal of LIF. This line wassubjected to two rounds of gene targeting aimed at the LIFR locus. Thetargeting vectors employed used the same homology arms as previouslydescribed (Li, Sendtner & Smith, 1995) but the selectable markercassette was replaced with an IRESBSDrpA and an IREShphpA cassette forthe first and second rounds of gene targeting, respectively. Theresultant cell line (LRK1) retained a high supertransfection efficiencyand was no longer responsive to LIF or other cytokines which can directES cell self-renewal via stimulation of LIFR. LRK1 cells were routinelymaintained in the presence of IL6 and sIL6R, which allowed stimulationof ES cell self-renewal via gp130 (Yoshida et al., 1994).

Library Construction

Co-cultures of γ-irradiated primary mouse embryonic fibroblasts and EScells (ZHTc6 cells; Niwa et al 2000) were initiated and maintained instandard ES cell medium lacking cytokines. The number of cells in theco-cultures was such that the MEFs formed a confluent monolayer and theES cells were seeded such that they would be in excess of the MEFs after3 days of growth. Staining of a representative plate for Alkalinephosphatase (a marker of ES cells) allowed estimation of the numbers ofES cells present at the time of cell lysis. This gave a ratio of ESRNA/MEF RNA of 12:1. RNA was prepared from 20 F180 flasks of cells andpolyadenylated RNA was prepared. RNA quality was monitored by examininga Northern blot for LIFR transcripts; only RNA producing a sharp 11 kbband with no detectable degradation was used further. cDNA wassynthesized from polyadenylated RNA by oligo d(T) priming using reagentssupplied in the Superscript plasmid system for cDNA synthesis andplasmid cloning (Life Technologies). cDNA was fractionated on apolyacrylamide gel and after recovery of cDNA>1 kb by electroelution,cDNA was ligated into Xhol/Notl digested pPyCAGIP. A library of 7.4×10⁵primary recombinants was produced after electroporation of E. colistrain DH10B. DNA was prepared from bacteria after overnight growth on15 cm diameter petri dishes seeded with 5×10⁴−10⁵ colonies each.

Library Screen

DNA (25 μg) from either library or empty vector was introduced into LRK1cells (6×10⁶) by electroporation and cells were seeded at 10⁶/9 cm dishor 5×10⁴/9 cm dish and cultured in IL6/sIL6R. After 2 days, medium waschanged to include puromycin and the cytokine was withdrawn from thehigh density plates. Cytokine was maintained on the low density platesto monitor transfection efficiency. Medium was changed every 2 daysuntil 9 days post transfection. At this time there were no cells aliveon the mock transfected plates and the plates transfected with emptyvector alone contained only differentiated cells. In contrast, some ofthe plates transfected with library DNA contained colonies whichappeared morphologically to resemble undifferentiated cells.Extrachromosomal DNA was therefore prepared from the cells on theseplates and transferred to E. coli DH10B. DNA was prepared from pools of10⁴ bacterial colonies and this was re-introduced into LRK1 cells andthe selection process described above repeated. Of 10 pools screened 2appeared positive and extrachromosomal DNA was prepared from these 14days post-transfection. DNA from one of these pools was further examinedas detailed below. After transfer to E. coli DH10B, DNA from a pool ofapproximately 250 colonies was prepared, re-introduced into LRK1 cellsand the selection process repeated. DNA was prepared from cells 14 or 19days after transfection. These were transferred to E. coli DH10B and 12miniprep DNAs prepared from the DNA harvested at 14 days posttransfection and 9 minipreps prepared from the DNA harvested at 19 dayspost-transfection. These DNAs were sequenced using an oligonucleotidelocated upstream of the 5′ cloning site.

Confirmation of Results

Sequence analysis of the 9 DNAs prepared 19 days post transfectionindicated that one of these plasmids contained a partial cDNA, one gavean uninterpretable sequence and 2 gave the same sequence which matched afull length sequence in the Genbank database. Four of the remaining 5plasmids lacked a cDNA insert and contained deletions to within 2 basepairs of each other within the IRES. The final plasmid lacked a cDNAinsert and had a deletion which removed a further 275 base pairs fromthe IRES. To confirm that some of these plasmids conferred theself-renewal activity a pool containing equal weights of all 9 plasmidswas prepared and transfected into LRK1 cells and subjected to theselection process described above. In contrast to cells transfected inparallel with an empty vector control, which all differentiated, cellstransfected with the pool of 9 plasmid DNAs exhibited anundifferentiated morphology. To determine which of the 9 plasmidsconferred the self-renewal activity individual plasmids was transfectedinto LRK1 cells and the selection process described above repeated.Plasmids carrying the partial cDNA, the uninterpretable sequence as wellas representatives of either of the 2 classes of deletion within theIRES failed to confer self-renewal activity. In contrast both of the twoplasmids which matched the full length sequence in the Genbank databasere-capitulated the self-renewal phenotype. This was judged bothmorphologically and by sustained expression of the marker alkalinephosphatase for 10 days.

Sequence analysis of the 12 DNAs prepared 14 days post-transfection,showed that 4 of these matched the same sequence as the 2 plasmidsdetailed above. One of these was transfected into LRK1 cells andpossessed the ability to confer cytokine-independent self-renewal asjudged by the maintenance of an undifferentiated colony morphology,sustained expression of alkaline phosphatase for at least 14 days andsustained Oct4 expression for at least 8 days. Furthermore, a comparisonof transfections of the PDF cDNA with a similar plasmid in which thecDNA sequences were restricted to the open reading frame showed PDFactivity to lie within the ORF.

EXAMPLE 2 Reversible Expression of a Pluripotency Determining Factor(PDF) Transgene

In order to demonstrate that the acquisition of a cytokine independentpluripotential phenotype caused by expression of PDF in ES cells wasreversible a transgene was made in which the transcription unit encodingPDF could be excised by site-specific recombination.

As a starting point, a plasmid encoding PDF identified in the initiallibrary screen was modified by placing a loxP site between the promoterand the translation initiation codon of PDF and a second loxP site afterthe polyadenylation signal. The downstream loxP site was followed by thecoding sequence of a GFP indicator gene and a polyadenylation signal. Inthis way, cells which had excised the loxP flanked PDF gene could beidentified by their altered fluorescent colour (see FIG. 2)

The plasmid encoding PDF was linearised in the vector backbone andtransfected into germ-line competent E14Tg2a cells. Cells were selectedfor growth in medium lacking gp130 stimulating cytokines. Individualclones were checked for green fluorescence and non-fluorescent cloneswere expanded in the absence of cytokine for 2 weeks. To eliminate theself-renewing effects of juxtacrine LIF family members, the LIFantagonist hLIF-05 was added to the medium during this period.Thereafter, the culture was split in two. The first of these cultureswas maintained as before while the other was maintained in LIF andtransfected with a plasmid encoding Cre (pCAGGCreGS). After 2 days, thecells were plated at low density and individual colonies examinedmicroscopically. Green fluorescent colonies were expanded and theirgenomic DNA analysed to confirm excision of transgenic PDF sequences.

Cells carrying both the floxed PDF gene or the excised transgene werethen injected into blastocysts separately and the recipient blastocyststransferred to foster mothers. At various times thereafter, embryos weredissected and analysed. Fluorescence microscopy allowed determination ofthe contribution of cells carrying the excised transgene to fetaltissues of all three germ layers. Some chimaeras were allowed to be bornand subsequently mated to demonstrate germline transmission from themanipulated ES cells.

EXAMPLE 3 Analysis of the Expression of PDF mRNA in Pluripotent Cells

Northern blotting analysis demonstrates that PDF mRNA is expressed in ECand ES cells but not in cell lines which are not pluripotent such asNIH3T3 fibroblast cells and B9 myeloid cells. Moreover, PDF expressiondecreases when ES cells are induced to differentiate by addition ofretinoic acid to the cultures. In situ hybridisation experimentsindicate that PDF mRNA is present in the inner cell mass of embryos atday 3.5 of development but not in later blastocysts nor in egg cylinderstage embryos. Therefore, expression correlates with cells which areeither themselves established pluripotent stem cell lines or areembryonic pluripotent cells from which stem cells can be established.

Thus, analysis by Northern blotting confirmed that PDF mRNA is expressedin ES cell/MEF co-cultures from which the cDNA library was synthesised(FIG. 4A). This analysis also confirmed hybridisation of the PDF cDNA tosmall B2 transcripts. The B2 hybridisation was eliminated when a proberestricted to the PDF ORF was used. Therefore, subsequent Northern andin situ analyses utilised the ORF probe. Expression was not detected inMEFs alone, nor was there any evidence of induction of PDF expression infibroblasts co-cultured with ES cells (FIG. 4B). This indicates that theES cells in the co-culture were the source of PDF cDNA. Analysis ofseveral cell lines indicated that PDF expression was highly restricted,being undetectable in parietal endoderm, yolk sac, fibroblast andhaematopoietic cells (FIG. 4C). Expression was detected in ES cells, EGcells and in both LIF dependent (PSA4) and LIF independent embryonalcarcinoma cell lines.

Examination of the human orthologue of PDF showed the corresponding mRNAto be expressed in an embryonal carcinoma cell line but not in alymphoid cell line (FIG. 4E). Several adult mouse tissues were surveyedfor PDF mRNA but no expression was detectable by Northern hybridisationof total RNA (FIG. 4F). The expression of PDF mRNA during ES celldifferentiation was next analysed. Whether through induceddifferentiation by retinoic acid or 3-methoxy-benzamide treatment, thelevel of PDF mRNA was rapidly reduced (FIG. 4D). Examination by in situhybridisation of ES cell cultures in which the LIF level was reduced inorder to allow the formation of colonies with a mixture ofdifferentiated and undifferentiated cells showed that PDF expression wasrestricted to the undifferentiated cells (FIG. 4B).

Pdf mRNA is found in pluripotent ES and EG cells and also in both mouseand human EC cells, but appears to be absent in other types of cell line(FIG. 4 and data not shown). Pdf expression is down-regulated earlyduring ES cell differentiation consistent with an intimate associationwith pluripotent stem cell identity.

EXAMPLE 4 Maintenance of Pluripotent Human Embryonic Stem Cells in theAbsence of Feeders/Feeder Extract

Human ES cells are cultured on a feeder layer of γ-irradiated mouseembryonic fibroblast (MEF) in 80% DMEM medium, supplemented with 100units/ml LIF, 1 mM glutamine, 0.1 mM 2mercapto-ethanol, 1% nonessentialamino acids, 4 ng/ml basic fibroblast growth factor and 20% KnockOut SR(a serum-free formulation) (GIBCO-BRL).

The plasmid shown in FIG. 1 (as well as a similar plasmid in which themouse PDF sequence is replaced by a human PDF sequence) is linearised inthe vector backbone and transfected into human ES cells. Thetransfection utilises the ExGen 500 method described in Eiges et al.except that the protocol is scaled up to use 10⁷ human ES cells. The dayafter transfection, the cells are trypsinised and split in two. One halfof the cells is plated onto a layer of Puromycin resistant MEFs at adensity of 10⁴/cm²; the other half is plated onto Puromycin sensitiveMEFs at a density of 10⁴/cm². Two days after replating, the cells on thePuromycin sensitive MEFs are trypsinised and plated onto gelatinisedplastic dishes at 10⁴/cm². Puromycin is added to both sets of culturesand medium replaced every 3-4 days. After approximately 14 days severalof the Puromycin resistant human ES cell colonies growing on feeders aretested for their ability to grow in the absence of a feeder layer bytrypsinisation and direct plating onto gelatin coated plates.

Puromycin resistant ES cell lines are assessed for their ability to beserially passaged and subcloned in the absence of a feeder layer overthe course of 6 weeks.

The differentiation potential of these cells is then tested (Eiges etal. 2001) following excision of the floxed PDF expression cassette asdescribed.

EXAMPLE 5 Characterisation of the PDF cDNA

A search for recognisable domains in the PDF sequence using SMART(http:H/smart.embl-heidelberg.de (Letunic et al., 2002; Schultz et al.,1998)) revealed the presence of a homeodomain between amino acidresidues 96 and 155, with no other obvious relationship to previouslycharacterised proteins. Further analysis by BLAST revealed that PDF wasmost closely related to several members of the NK2 family. However, inno case was this identity greater than 50% (FIG. 3). Therefore, PDF iseither the founding member of a novel homeodomain family or is a uniquevariant homeodomain (Shashikant et al., 1991).

A comparison of PDF with the most closely related human sequencerevealed overall identity of 60%. Sequence conservation was mostpronounced over the homeodomain where the two sequences were 87%identical (FIG. 3B). This far exceeds that the level of identity seenbetween the homeodomains of PDF and other mouse proteins, indicatingthat this is the human orthologue of PDF. Of the 8 non-identical aminoacids 6 are located in the N-terminal arm around a-helix 1, regions ofthe homeodomain considered to be more loosely associated with the DNAtarget. Outwith the homeodomain there are 4 regions in which acontiguous stretch of more than 4 amino acid residues is conserved. Ofthese conserved regions only one, a serine rich motif, is N-terminal tothe homeodomain, the remainder lying C-terminal to the homeodomain. Thehuman sequence contains an insertion of seven amino acids between thefirst and second of these C-terminal identity motifs. In the mousesequence, between the homeodomain and the C-terminal conserved sequencesthere is a 46 amino acid stretch in which every fifth residue istryptophan. Moreover, within this sequence the first 31 amino acidsrepresent a simple reiteration of the sequence WnsQTWTNPTW (n=G,S,N ands=S,N). The conservation of the sequence in the human is less strikingand contains a short deletion relative to the mouse sequence. Yet withthe exception of the 16^(th) residue every fifth residue remainstryptophan.

In the 3′UTR of the PDF mRNA there is a B2 repetitive element orientedin the opposite transcriptional direction to PDF (FIG. 3C). Whether thissequence is transcribed by RNA polymerase III is not clear although thesequence of the split promoter suggests that it may be (Galli et al.,1981). Since B2 elements are expressed at high levels in embryonic cells(Ryskov et al., 1983) it is possible that the presence of the B2 withinthe 3′UTR of PDF mRNA may contribute to the regulation of PDF geneexpression either directly through transcriptional interference or bymore indirect means.

EXAMPLE 6 Analysis of hPDF Function in Mouse ES Cells

The ability of the human PDF orthologue to replicate the activity ofmouse PDF was tested by placing the human ORF in the pPyCAGIP vector andtransfecting mouse ES cells. This shows that the human sequence wascapable of directing cytokine independent self-renewal of mouse ES cells(FIG. 3). This activity was reduced compared to mouse PDF, presumablydue to the considerable sequence divergence. The specificity of theeffect of PDF was tested by a similar experiment in which the ORF of oneof the mouse homeobox genes most closely related to PDF (Nkx2.5) wasexpressed in ES cells. In this case no cytokine independent self-renewalwas evident; in fact the resultant cells appeared morphologicallydifferentiated even in the presence of LIF. This indicates that cytokineindependence is specifically conferred by PDF and is not a commonattribute of homeodomain proteins.

EXAMPLE 7 In Vivo Expression of mPDF

We examined distribution of PDF mRNA in vivo (FIG. 5). No expression isseen during early cleavage stages. The first sign of PDF mRNA expressionis in compacted morulae. It is striking that the hybridisation signal islocalised to interior cells, the future inner cell mass. In the E3.5blastocyst expression is confined to the cells of the inner cell massand is absent from the trophectoderm. In later blastocysts PDF mRNA isfurther restricted to the epiblast and is excluded from the primitiveendoderm. PDF transcripts appear as a temporal wave with maximal levelsbetween the late morulae and the mid blastocyst. In blastocysts about toimplant, the PDF mRNA level has dropped below the level ofvisualisation. Significantly, however, transcripts remained detectablein the epiblast of blastocysts in diapause. We also examined PDFexpression in primordial germ cells as these can be converted intopluripotent EG cells (Matsui et al., 1992). Expression is not evident inthe migratory germ cells at E8.5 but PDF mRNA is readily detectable inthe genital ridges of E11.5 embryos in a pattern indicative oflocalisation to the primordial germ cells (FIG. 5B).

EXAMPLE 8 Relationship of PDF with Other Known Mediators of Pluripotency

The dose response of ES cells to increasing levels of PDF was assessedby episomal expression in LRK1 cells. This was achieved by transfectionof episomal constructs directing differing levels of cDNA expression(FIG. 8A). The proportion of the resulting colonies that couldself-renew in the absence of cytokine correlated with the level ofexpression expected from the episomes based on similar studies measuringgfp expression levels (data not shown). Furthermore, as observed forcells carrying integrated PDF transgenes, the addition of LIF tocultures of cells carrying PDF episomes augments self-renewal asassessed both in terms of the number of self-renewing colonies formedand also colony morphology. Essentially identical results were obtainedby transfection of the same DNAs into the CCE derived polyoma large Texpressing cell line MG1.19 (Gassmann et al., 1995).

We investigated whether the co-operative effect of the gp130 signal andPDF overexpression is due to the PDF gene being a transcriptional targetof STAT3. Using chimaeric receptors in which the intracellular portionof gp130 is linked to the extracellular portion of GCSF-R it is possibleto investigate STAT3 targets in ES cells. An analysis of the eight knownSOCS family members, which are all potential STAT targets, indicatedthat only SOCS3 is significantly stimulated in ES cells by LIF (FIG. 8Band data not shown). This stimulation does not occur when the four STATbinding sites in the receptor are prevented from binding STAT3 bymutation from tyrosine to phenylalanine (FIG. 8B). In contrast, SOCS3induction is enhanced and sustained when the negative regulatorytyrosine is similarly mutagenised, a result consistent with thesustained STAT3 activation observed in these cells (Burdon et al.,1999). In contrast to the situation with SOCS3, no induction of PDFexpression is evident upon LIF stimulation nor upon G-CSF stimulation ofcell expressing chimaeric receptors (FIG. 8B).

EXAMPLE 9 Relationship of PDF with Oct 4

The ability of PDF to substitute for Oct4 in self-renewal of ES cellswas then tested using ZHBTc4.1 cells. These cells carry two null allelesfor Oct4 but their self-renewal can be sustained due to the presence ofa doxycycline responsive Oct4 transgene (Niwa et al., 2000). When theexpression of the Oct4 transgene is repressed by administration ofdoxycycline, the cells differentiate. ZHBTc4.1 cells were transfectedwith linearised pPyCAGIP sequences carrying no insert, Oct4 or PDF. Aspreviously documented, this Oct4 plasmid prevents the differentiationcaused by doxycycline induced repression of the Oct4 transgene (Niwa etal., 2002). However, the PDF expression vector could not (FIG. 8C).Therefore, although PDF can sustain the ES cell phenotype in the absenceof gp130-mediated gp130 stimulation, it cannot do so in the absence ofOct4.

EXAMPLE 10 ES Cell Identity is Faithfully Maintained by PDF Expression

We investigated the consequences of PDF transfection in ES cells that donot contain polyoma LT. A loxP containing construct was employed suchthat the PDF cDNA could subsequently be excised by Cre recombinase.Following isolation of stable integrants, site specific recombinationcan then be used to remove the PDF ORF and simultaneously bring GFPunder CAG promoter control (FIG. 6A). The excision of the PDF transgeneallows assessment of any genetic or epigenetic changes to the cellscaused by temporary transgene expression. In these experiments we aimedto test the ability of cells overexpressing PDF to be propagatedclonally in the absence of gp130 signalling. We wished to examine twoissues; firstly, whether enforced PDF expression preventeddifferentiation in a range of circumstances and secondly, whetherpluripotency and embryo colonisation capacity could be sustained in theabsence of gp130 signalling.

Clonal transfectants were isolated by selection in puromycin andexpression of PDF transgene mRNA was confirmed by Northern analysis(FIG. 6B). Cells were seeded at low density and expanded in the presenceof the LIF antagonist, hLIF-05 (Vernallis et al., 1997) for at least 7days through two passages. hLIF-05 blocks the ability of all known LIF-Rligands to engage the LIF-R/gp130 complex. Parental cells treated inparallel produced only differentiated cells that failed to expand. Incontrast, lines containing the floxed PDF-IRES-pac cassette maintainedan undifferentiated morphology and continued to proliferate.

Subsequently, LIF was added and cells were transiently transfected withCre. Colonies in which the PDF expression cassette had been eliminatedwere identified by expression of GFP and a restoration of puromycinsensitivity. This procedure was performed using two independent ES celllines E14Tg2a and ZIN40. Chromosomal spreads were examined for severalof the resulting clones and in all cases were found to be predominantly40XY.

Colony forming assays were performed as a rigorous assessment of thephenotype of PDF transfectants and their Cre-treated derivatives. Cellswere plated at clonal density in medium supplemented with LIF, withoutLIF or with the LIF antagonist hLIF-05. After culture for 6 days, plateswere stained for alkaline phosphatase activity and the proportion ofcolonies consisting solely of undifferentiated cells quantitated (FIG.6C). Neither the parental nor the GFP expressing cells formed any fullyundifferentiated colonies in the absence of LIF or in the presence ofthe LIF antagonist. PDF transfectants, however, generated appreciablenumbers of such pure stem cell colonies. Upon addition of LIF thisproportion rose to >80% of colonies. Following Cre mediated excision ofthe transgene this enhanced response to LIF was also lost.

The difference in the proportion of cells expressing the PDF transgeneforming fully undifferentiated colonies in the presence or absence ofLIF is accompanied by a change in the morphology of the colonies (FIG.6D). In both cases the colonies are composed of small undifferentiatedcells with a scant cytoplasm and prominent nucleoli. Without LIF, thePDF expressors grow as a monolayer and are essentially indistinguishablefrom parental or Cre-treated derivative ES cells cultured in thepresence of LIF. The combination of LIF plus PDF expression, however,causes the colonies to adopt a tightly compacted morphology in which thecells preferentially adhere to one another rather than grow out over thesubstratum. This resembles the “classical” appearance of ES cellscultured on feeder layers.

Forced expression of PDF allows ES cells to self-renew in the absence ofgp130 stimulation, culture conditions in which the parental cellsdifferentiate. We next determined whether PDF expressor cells wouldself-renew or differentiate when exposed to agents that normally causedifferentiation. When cultured for four days following exposure to3-methoxybenamide (MBA) or all-trans retinoic acid (RA), PDF expressingcells retained an undifferentiated appearance, whereas cultures of boththe GFP derivatives and the parental cells underwent morphologicaldifferentiation (data not shown). These morphological features arereflected at the molecular level in continued expression of Oct4 by PDFexpressors compared with dramatic down-regulation in Cre derivatives(FIG. 6E). These data establish that PDF transfectants are refractory tocues that normally direct differentiation in monolayer cultures but thatfollowing transgene excision differentiation capacity is restored.

The ability of PDF expressing cells to respond to cellular interactionsthat induce differentiation was then investigated by aggregation insuspension culture, conditions that lead to formation ofmultidifferentiated embryoid bodies from parental ES cells (Doetschmanet al., 1985). After eight days in aggregation culture, embryoid bodieswere plated singly per well of a 48 well dish and subsequently scoreddaily for spontaneous contractions, an indicator of cardiomyocytedifferentiation. FIG. 7A shows that the incidence of cardiogenesis isreduced by approximately 50% in PDF expressors relative to their Crederivatives. Furthermore, the emergence of cardiac cells from PDFexpressors is retarded relative to their Cre derivatives. In fact thegraph in FIG. 7 underestimates the difference between the two classessince wells derived from cells expressing the floxed transgene containedonly small beating areas whereas wells derived from the Cre derivativecells had extensive beating areas, in some cases covering the wholeoutgrowth. Embryoid bodies were also exposed to retinoic acid for thelast 4 days of aggregation culture and plated out under conditionsfavouring neuronal differentiation (Bain et al., 1995; Li et al., 1998).E14Tg2a cells formed large numbers of cells of neuronal morphology thatexpressed neuron-specific type III b-tubulin (TuJ) (FIG. 7B). Thecontinuous presence of LIF inhibited differentiation of TuJ positivecells. Emergence of TuJ positive cells was also blocked in culturesderived from ES cells expressing the floxed transgene, but was restoredin their Cre derivatives. However the appearance of the cells remainingin the wells from the E14Tg2a (+LIF) and EF4 cultures was not identical.The former contained many cells with the morphology of ES cells, whereasthe PDF expressors had a more dispersed appearance suggesting that theycould be partly differentiated. These observations indicate that EScells expressing PDF do not undergo differentiation efficiently inmulticellular aggregates environment but that some degree ofdifferentiation is still induced. Most significantly, the Cre derivativecell lines appear unaffected by their period of clonal expansion drivenby enhanced PDF expression since they possess comparable potential tonormal ES cells for differentiation under all conditions examined.

Finally, we investigated whether forced expression of PDF circumventsthe requirement for LIF-R/gp130 mediated signalling in maintenance ofembryo colonisation properties. PDF transfectants (isolated in theabsence of LIF and then cultured in the presence of LIF antagonist forover 1 week) from which exogenous PDF sequences had been removed by Cremediated excision, were injected into mouse blastocysts. To date we haveassessed Cre derivative lines from one of the PDF transfectants.Examination of the resulting chimaeras at mid-gestation demonstratedwidespread contribution of the cells to tissues of the developingembryos as revealed by GFP expression (FIG. 7D). Similarly injectedembryos were brought to term and resulted in the production of livechimaeras (Table 1).

These results establish: (i) that cells expressing the PDF transgeneself-renew in a cytokine independent manner; (ii) that LIF actsco-operatively with the PDF transgene to confer enhanced self-renewalcapacity; and, (iii) that the effects of PDF transgene expression arefully reversible following transgene excision.

TABLE 1 Live born chimaeras obtained from Cre derivatives of PDFtransfectants Line Mice born Chimaeras EF1C1 7 3 EF1C3 21 10 EF1C5 11 11

EXAMPLE 11 Expression of PDF cDNA in a Human Pluripotent Cell Line

In this study, we have demonstrated the factor-dependent maintenance ofpluripotentiality following expression of the human cDNA forPluripotency Determining Factor (hPDF) in a human pluripotent EC cellline. This clonally derived cell line, Germ Cell Tumour 27X-1(GCT27X-1), kindly provided by Assoc. Prof. Martin Pera, MonashInstitute for Reproduction and Development (MIRD), Melbourne, Australia)has been previously shown to give rise to yolk sac, trophoblast, skin,cartilage, glandular epithelium, neurectoderm and other tissuesrepresentative of the three primitive germ layers in xenografts. Thesame cell line also shows spontaneous differentiation in vitro intosomatic and extraembryonic cell types (Pera et al., 1998). The GCT 27X-1cell line is routinely maintained either on a layer of mitoticallyinactivated mouse STO feeder cells (FIG. 9 a) or in the absence offeeder cells with the addition of an uncharacterised factor secretedfrom the feeder-free culture of a human yolk sac carcinoma-derived cellline, GCT44 (kindly provided by Martin Pera, MIRD, Melbourne,Australia), (FIG. 9 b). The withdrawal of this conditioned medium fromlow and clonal density GCT 27X-1 cell cultures results in their completedifferentiation (FIG. 9 c).

The hPDF transcription factor is endogenously expressed in pluripotentGCT 27X-1 hEC cells (FIG. 10) and is therefore postulated to play a rolein the maintenance of self-renewal in this and other human pluripotentcell lines.

Here we demonstrate the loss of pluripotentiality in GCT 27X-1feeder-free cell cultures in response to the withdrawal of the yolk saccarcinoma cell-derived conditioned medium and the maintenance ofpluripotentiality at a clonal level where exogenous hPDF is expressed.

These findings show that the hPDF transcription factor provides a basisfor robust culture systems for human ES cells.

Experimental Strategy

A construct containing a human cDNA sequence encompassing the openreading frame for the hPDF gene was transfected both by lipofection andelectroporation techniques into the GCT 27X-1 cells. Stable transfectantcolonies were established in the absence of feeder cells, with andwithout the addition of GCT 44-derived conditioned medium required forthe support of hEC cell growth and maintenance. Stable colonies wereselected in the presence of the puromycin antibiotic and those showingthe maintenance of a tight hEC phenotype (as judged by morphology) werepicked and expanded from both conditions. These clonal hPDF-expressingcell lines were subsequently tested at both routine and clonal densityfor their ability to maintain a pluripotent phenotype in the absence ofconditioned medium, with wild type GCT 27X-1 cells and GCT 27X-1 cellstransfected with a similar construct but lacking the hPDF sequence ascontrols.

Definitive undifferentiated hEC cell phenotype in PDF-expressing cellswas confirmed by indirect immunofluorescent staining for the markerCluster Designation 30 (CD30), a member of the Tumour Necrosis Factorreceptor superfamily. CD30 is a specific marker of human EC cells and isdown-regulated during in vitro differentiation (Latza et al., 1995; Peraet al., 1997). There is no evidence for the presence of CD30 on mouse ECor mouse ES cells.

This experimental strategy is outlined in flow chart form (FIG. 11).

Materials and Methodology Vector Constructs

An 8.8 kb construct, termed Floxed hPDF (FIG. 12 a), contains the openreading frame for the hPDF cDNA (˜900 bp) operating under a humancytomegalovirus immediate early enhancer (CMV IE) and the human actinpromoter, upstream of a bicistronic reporter cassette providing forIRES-mediated expression of the puromycin resistance gene, allencompassed within loxP recombination sites. In addition, the vectorcontains the enhanced GFP reporter gene downstream of the loxP sites,providing for visualisation by fluorescence where Cre-mediatedrecombination occurs.

A similar non-floxed construct pPyCAGegfpIP (“GFP control” vector),(FIG. 12 b), in which the hPDF sequence in the Floxed hPDF vector isreplaced by the enhanced GFP reporter gene upstream of theIRES-puromycin reporter cassette, was used as a control to provide “nonhPDF”-expressing transfected hEC cells for each experiment.

hEC Cell Culture

GCT 27X-1 cells were routinely cultured on a feeder layer of mitoticallyinactivated mouse STO cells in a 1:1 v/v αMEM/Ham's F-12 medium(Invitrogen, Groningen, The Netherlands) supplemented with 2 mML-Glutamine (Invitrogen), 2.7 g/L sodium bicarbonate (Sigma ChemicalCo., St. Louis, Mo., USA) and 10% FCS (Commonwealth Serum Laboratories,CSL, Melbourne, Australia), and maintained at 37° C. with 5% CO₂. Cellswere passaged each 7 days using digestion with a 5 mg/ml dispasesolution (Sigma) prepared in the above medium, and replated at a densityof no less than 1/10^(th) of the harvest.

For transfection experiments and clonal assays, GCT 27X-1 cells werecultured without STO feeder cells but with the addition of conditionedmedia obtained from the feeder-free culture of a human yolk saccarcinoma-derived cell line CGT 44 (cells kindly provided by MartinPera, MIRD, Melbourne, Australia) at 25% v/v in the supplementedαMEM/Ham's F-12 medium described above. Single cell suspensions wereprepared by trypsin digestion with a 0.025% trypsin (Invitrogen)/1 mMEDTA (Sigma)/1% chicken serum (Invitrogen) solution prepared in PBSbuffer. Clonal density cell cultures were seeded at 200-300 cells/cm²and low density cultures at 5000 cells/cm².

The GCT 44 cell line was routinely maintained on mitotically inactivatedmouse STO feeder cells in high glucose DMEM medium (Invitrogen)supplemented with 2 mM L-Glutamine (Invitrogen), 3.7 g/L sodiumbicarbonate (Sigma) and 10% FCS(CSL), and maintained at 37° C. with 5%CO₂ but passaged by digestion with a 0.25% trypsin (Invitrogen)/1 mMEDTA (Sigma) solution prepared in PBS buffer. Conditioned medium wascollected on days 7, 10 and 14 from feeder-free cultures, filtersterilized and stored at 4° C. for up to 2 months. Each batch ofconditioned medium was tested for the ability to maintain feeder-freeclonal cultures of GCT 27X-1 hEC cells as described above (data notshown).

The mouse STO feeder cells (kindly provided by Martin Pera, MIRD,Melbourne, Australia) were routinely maintained in high glucose DMEMmedium (Invitrogen) supplemented as above for GCT 44 culture. RoutineSTO cultures were passaged twice-weekly by digestion with a 0.25%trypsin (Invitrogen)/1 mM EDTA (Sigma) solution prepared in PBS buffer.Mitotic inactivation of STO cells was achieved by incubation of aroutine culture for 2-3 hours in a 16 μg/ml Mitomycin-C solution (Sigma)prepared in supplemented DMEM medium (as above) and washed twice withPBS prior to trypsinisation.

For routine morphological examination of hEC cells, cultures were fixedand stained with Leishman's as previously described (O'Brien, 2001).

Cell cultures were routinely inspected by phase-contrast microscopyusing either Leica DMIL (Leica Microscopy Systems, Wetzlar, Germany) orNikon Diaphot 300 (Nikon Inc., Melville, N.Y., USA) microscopes.Fluorescing control GFP cell cultures were examined on a Nikon Diaphot300 microscope using a 470/40 nm Nikon filter. Fluorescent data wascaptured on a Leica DFC300 F imaging system. All cell culture wasperformed using tissue culture grade plasticware supplied by Falcon(Becton Dickinson, Lincoln Park, N.J., USA) or Nunc (Roskilde, Denmark).

Stable Integrative Transfection of hEC Cells

The floxed hPDF and GFP control vectors were each linearised by standardPvu/restriction enzyme digestion and transfected into GCT 27X-1 cells byboth electroporation and lipofection strategies.

Electroporation: A single cell suspension of 20-30×10⁶ hEC cells wasprepared by trypsin digestion and transfected with 40 μg of linearisedDNA using a BTX ECM830 square wave electroporator (BTX, San Diego,Calif., USA), set to deliver an electrical discharge of 0.8 kV and atime constant of 0.1 ms (3 μF capacitance).

Lipofection: 2×10⁶ hEC cells were seeded to a 78.5 cm² culture dish oneday prior to transfection. Lipofection was performed by incubation ofthe cells in serum free medium for 6 hours with Lipofectamine 2000reagent (Invitrogen) at 60 μg (1 μg/μl solution) and 20 μg of linearisedDNA, according to the manufacturer's instructions. Cells weretrypsinised 48 hours post-lipofection and plated at the same densitiesas for electroporated cells.

Transfected cells and control untransfected cells were plated at adensity of 2×10⁶ cells in 78.5 cm² dishes (˜25,500 cells/cm²) andcultured for up to 15 days both with and without conditioned medium,refreshing medium each 3-4 days. Puromycin selection was applied 2-3days following transfection, initially at 0.4 μg/ml then at 1.0 μg/mlfrom days 10-15 post-transfection for effective selection of stableintegrants. Puromycin resistant hEC colonies were picked into 2.0 cm²wells and expanded for subsequent freezing and further analyses. Toensure maintenance of self-renewing GFP control hEC cell lines, onlycolonies from conditioned medium plates were picked and expanded. Allprocedures for achieving stable integrative transfection of hEC cellswere essentially as previously described for mouse ES cells (O'Brien,2001).

CD30 Immunostaining

hEC cell cultures were prepared for indirect immunofluorescent stainingwith a mouse anti-human CD30 monoclonal antibody (Dako, Glostrup,Denmark) by washing twice with PBS buffer, fixing in ethanol for 10minutes on ice and storing at −20° C. Staining was performed using a1:20 dilution of the primary antibody in a 0.1% Triton X-100 (Sigma)/2%goat serum (Invitrogen)/13.3% v/v BSA (Invitrogen) staining diluentprepared in PBS buffer. A Cy™3-conjugated goat anti-mouse IgG secondaryantibody (Jackson ImmunoResearch Laboratories Inc., Westgrove, USA) wasused at a 1:200 dilution in the above diluent buffer. CD30 positivecells were viewed using a 535/50 nm Nikon filter and comparative Hoechst(Sigma) staining with a UV 330-380 nm Nikon filter. Fluorescent data wascaptured on a Leica DFC300 F imaging system. All procedures forimmunofluorescence staining were as described in SCS Ltd laboratoryprotocols.

Northern Blot Analysis

Poly A+ mRNA was prepared from subconfluent cultures of GCT 27X-1 cellscultured both on STO feeder cells and without feeder cells but in thepresence of conditioned medium as described above. In addition, poly A+mRNA was prepared from routine cultures of STO cells and E14Tg2a mouseES cells, as controls for subsequent hybridisation. RNA lysates wereprepared directly from cell cultures and poly A+ mRNA isolated byaffinity chromatography on oligo(dT) cellulose (New England Biolabs,Beverly, Mass., USA) as previously described (O'Brien, 2001).

Extracted mRNA samples (3-3.5 μg) were electrophoresed on a 1% w/vagarose/0.66 M formaldehyde denaturing gel run in 1.times.MOPS buffer.RNA was transferred to Hybond N+ charged membranes (Amersham PharmaciaBiotech, Amersham, Buckinghamshire, UK) and hybridised with a³²P-labelled (Amersham Pharmacia Biotech) 960 by hPDF cDNA fragmentisolated by Eco RI restriction enzyme digestion from the pPyCAGhPDFIPvector (vector kindly supplied by Ian Chambers, ISCR, Edinburgh, UK). An800 by Hind III/EcoR I fragment of Gapdh coding sequence was used tore-hybridise some membranes to provide a loading control for mRNAsamples (plasmid DNA kindly provided by Kate Loveland, MIRD, Melbourne,Australia). Membranes were exposed to Biomax MS (Kodak, Cedex, France)and Hyperfilm MP (Amersham Pharmacia Biotech) X-ray films betweenintensifying screens at −70° C. for up to 24 hrs. Blotting,hybridisation and routine molecular procedures were performed asdescribed by Sambrook et al., (1989).

Results Self-Renewal and Differentiation of GCT 27X-1 Cells

Routine culture of the GCT 27X-1 hEC cells was established as describedin the methodology. Maintenance of a pluripotent phenotype wasdemonstrated, as judged by morphology, when cultured on a layer of STOfeeder cells (FIG. 9 a) or in the absence of STO feeder cells but in thepresence of GCT 44-derived conditioned medium, at both a routine (FIG. 9b) and a clonal density (FIG. 14 g). Withdrawal of conditioned mediumfrom feeder-free cultures of hEC cells demonstrated the initiation ofcell differentiation and complete loss of pluripotency in low densityand clonal density cultures (FIG. 9 c). It was further demonstrated thatwhile feeder-free hEC cultures could not maintain a pluripotentmorphology in cultures containing less than 25% (v/v) conditionedmedium, a replacement of the routine culture medium with more than 75%(v/v) conditioned medium could not wholly support the survival andgrowth of the cultures (data not shown).

Endogenous Expression of hPDF in hEC Cells

To confirm endogenous expression of the hPDF gene in pluripotent GCT27X-1 cells, a poly A+ mRNA Northern blot was prepared and hybridisedwith a radiolabelled probe corresponding to ˜900 by of hPDF cDNAsequence contained in the Floxed hPDF vector. Results in FIG. 10 showthat the hPDF probe strongly detects a hybridisation band of ˜2.4 kb inGCT 27X-1 cells grown both on feeder cells and without feeder cells butin the presence of conditioned medium. A second lesser hybridisingtranscript of ˜5.6 kb size was also detected for both cultures andconfirmed in repeat experiments on different RNA preparations (data notshown). No hybridising band was detected from mRNA prepared from aroutine STO cell-only culture. A weak hybridising band of ˜2.2 kb wasdetected in mouse E14Tg2a ES cell mRNA and is indicative of a homologousPDF transcript expressed in mouse pluripotent cells. Relative amounts ofpoly A+ mRNA in different gel lanes was determined by re-hybridisationof a membrane with the housekeeping gene Gapdh. This indicated that themouse PDF transcript was detected at much lower levels, in comparison tothe transcripts detected in hEC cells (data not shown).

The ˜2.4 kb transcript detected in GCT 27X-1 hEC cell mRNA correspondswith that observed previously for a nullipotent hEC cell line GCT 27C4derived from the same parental line.

Transfection of hPDF and Control GFP Vectors

In trial transfection experiments with the GFP control vector, bothelectroporation and lipofection strategies gave comparable results inGCT 27X-1 cells (data not shown). Transfection of GCT 27X-1 hEC cellswith the Floxed hPDF vector was performed by both electroporation andlipofection, with a comparative GFP control vector transfection done bythe lipofection method only (Table 2).

Stable integrative colonies harbouring the Floxed hPDF vector wereestablished as feeder-free cultures both in the presence and absence ofconditioned medium. Stable integrative colonies harbouring the GFPcontrol vector were established under the same conditions, for the samenumber of plated cells. Stable, moderate sized colonies were establishedin 10-15 days for both vectors, with puromycin control plates foruntransfected cells essentially clear of growth after this time.

Results in Table 2 indicate a slightly higher efficiency of transfectionof GCT 27X-1 cells by lipofection than electroporation for the FloxedhPDF vector, but appear to show no difference in the number of stablehPDF integrant colonies established in the presence of conditionedmedium to those without conditioned medium, compared with GFP controltransfectants. While the culture of transfected cells withoutconditioned medium does not appear to hinder the ability for colonies tobecome established, the morphology of these does however, differ forFloxed hPDF and GFP control transfectants, as discussed in the followingsection (see Table 3).

An equivalent number of primary colonies, all displaying a tightpluripotent hEC phenotype were picked and expanded from both conditionsfor the hPDF transfectants. While GFP control colonies were establishedwithout conditioned medium for observation of morphology in stablecolonies (see Table 3), these were not picked due to the anticipatedinitiation of differentiation in these colonies over this time frame.

It was noted that transfected hPDF cells appeared to take the sameamount of time to establish colonies either in the presence or absenceof conditioned medium, however, following picking those growing withoutconditioned medium expanded at a slower rate than those cultured withconditioned medium. This is a similar observation to that forPDF-expressing mouse ES cells established in cultures with and withoutLIF (see Example 8). It is also noteworthy that GFP control coloniesexpanded in conditioned medium displayed a slightly faster growth ratethan their hPDF counterpart.

Morphological Observations for hPDF and GFP Control Transfectants

Stable transfectant colonies established following transfection with theFloxed hPDF and GFP control vectors and cultured in the presence andabsence of conditioned medium were compared morphologically followingLeishman's staining (Table 3). Colonies were scored as “tight”, “medium”or “loose” to describe those puromycin resistant colonies maintaining atight pluripotent phenotype, those starting to differentiate whilemaintaining some hEC phenotype and those that were completely loose anddifferentiated, respectively (FIGS. 13 a-c).

Examination of colony morphology for primary transfectants, as shown inTable 3, indicates that while hPDF transfectants displayed a higherdegree of tight hEC morphology in conditioned medium compared with GFPcontrol transfectants, there was an increase in the ability for hPDFtransfectants to hold a pluripotent phenotype without conditioned mediumcompared with GFP control colonies—for which an increase in loose,differentiating phenotype was observed.

While the variation in morphology for transfectants resulting from eachvector may be influenced by the site of DNA integration into the genome,those resulting from the floxed hPDF transfection appear to display anincreased ability to establish and maintain a pluripotent phenotype,even more so under conditions that would normally initiatedifferentiation and loss of pluripotency in colonies.

Generally, colonies observed on those hPDF transfection plates culturedwithout conditioned medium were smaller in size than those growing inconditioned medium, albeit with many displaying a tight pluripotentphenotype. It also remains possible that a synergistic effect occurswhere hEC cells expressing exogenous hPDF are cultured in the presenceof conditioned medium.

Maintenance of Pluripotency in hPDF-Expressing Transfectants

The ability of hPDF-expressing stable transfectant lines to definitivelymaintain a pluripotent phenotype under conditions that normally initiateGCT 27X-1 cell differentiation was confirmed by indirectimmunofluorescent staining with the CD30 marker, specific to pluripotenthEC cells and not expressed in differentiating cell types.

Six hPDF-expressing cell lines, including one established and culturedwithout conditioned medium from the time of transfection, were culturedat a clonal density in both the presence and absence of conditionedmedium. Four of the GFP control hEC transfectant cell lines (maintainedin conditioned medium for self-renewal) as well as untransfected GCT27X-1 hEC cells (also maintained in conditioned medium without feedercells since the time of transfection experiments), were cultured inparallel at clonal density under the same conditions as for the hPDFlines. Morphological analysis and CD30 indirect immunofluorescentstaining was performed when GFP control and GCT 27X-1 cells culturedwithout conditioned medium demonstrated complete or significantdifferentiation of colonies. It was noted that the untransfected GCT27X-1 cells established expanded colonies at a faster rate than GFPcontrol and hPDF cell lines.

Cell lines expressing the hPDF cDNA were shown to maintain a pluripotentphenotype at clonal density, with and without conditioned medium, whilethe GFP control cells and GCT 27X-1 cells could only maintainpluripotency where conditioned medium was present in the culture.

Results in FIG. 14 show the morphology of two hPDF clones expanded fromtransfection plates in the continuous presence (hPDF clone D7) orcontinuous absence (hPDF clone E7) of conditioned medium, and culturedat clonal density with and without conditioned medium (FIGS. 14 a-d).Both hPDF D7 and E7 clones demonstrate the ability to establish andmaintain hEC colonies displaying a pluripotent cell phenotype under bothconditions. Parallel clonal cultures of a GFP control clone, C15, and ofuntransfected GCT 27X-1 cells, confirm the ability of these cells toonly establish and maintain pluripotent hEC colonies in the continuedpresence of conditioned medium. In the absence of conditioned medium theC15 and GCT 27X-1 cells are induced to differentiate, with few or no hECcells remaining in the loosely formed colonies (FIGS. 14 e-h).

Results in FIG. 15 confirm the above morphological observations withcomparative CD30 and Hoechst immunofluorescence staining of individualcolonies established from hPDF D7, hPDF E7, GFP C15 and GCT 27X-1 celllines, cultured in the presence and absence of conditioned medium. Allcell lines displayed the ability to maintain self-renewing CD30-positivecolonies when cultured in the absence of feeder cells and presence ofconditioned medium (FIGS. 15 a-h). As the GCT 27X-1 cells establishedlarge expanded colonies more rapidly than the transfected cells, many ofthese had started to differentiate at their periphery as would beexpected with overgrowth (FIGS. 15 g-h). In the absence of both feedercells and conditioned medium, only hPDF-expressing hEC cells were ableto maintain CD30-positive colonies, while GFP control and GCT 27X-1 hECcells could not maintain self-renewing colonies (FIGS. 15 i-p). Again,any residual CD30-positive cells observed in GCT 27X-1 colonies, couldbe attributed to the more rapid expansion and overgrowth of thesecolonies (FIGS. 15 o-p).

It was observed that the hPDF E7 cell line displayed a generally tighterhEC colony morphology (FIGS. 14 a-d; FIGS. 15 a, c, i, k) and strongerdetection of the CD30 marker (FIGS. 15 b, d, j, l) when compared withhPDF D7 cells, and may reflect the site of random DNA integration intothe genome in each clone. The hPDF E7 cell line was initiallyestablished in the absence of both STO feeder cells and conditionedmedium following transfection. After more than 8 weeks of continuousexpansion under these culture condition the hPDF E7 hEC cell linecontinues to display a phenotype of self-renewing hEC cells (FIG. 16).

This demonstrates that exogenous expression of the human PDF cDNA in ahuman pluripotent cell line results in the maintenance of a pluripotentphenotype under conditions that normally drive these cells todifferentiate and lose the ability to self-renew.

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TABLE 2 Stable integrative transfection of GOT 27X-1 cells with a hPDFvector. Pluripotent GCT 27X-1 cells were transfected with the FloxedhPDF vector by both electroporation and lipofection strategies andstable transfectant colonies established without STO feeder cells in thepresence and absence of conditioned medium (+/−CM), under puromycinselection. To provide control cell lines, GOT 27X-1 cells transfected bylipofection with the GFP control vector were established under the sameconditions. Equivalent numbers of colonies were picked and expanded fromall conditions, excepting GFP controls which were picked only fromconditioned medium plates to maintain hEC colonies. GFP Floxed hPDFvector control vector Electroporation Lipofection Lipofection # hECcells 23 × 10⁶ 2 × 10⁶ 2 × 10⁶ transfected seeded 24 hrs seeded 24 priorhrs prior # transfected  2 × 10⁶ 2 × 10⁶ 2 × 10⁶ cells seeded per plated48 hrs plated 48 hrs 78.5 cm² plate post-t/f post-t/f stable 1:5102(0.020%) 1:3597 (0.028%) 1:3425 (0.029%) integrants 392  556  584established in +CM stable 1:4902 (0.020%) 1:3125 (0.032%) 1:2809(0.036%) integrants 408  640  712 established −CM # primary 24 24  24colonies picked: +CM culture # primary 24 24  0 colonies picked −CMculture

TABLE 3 Morphology of stable integrative hPDF and control GEPtransfectant GCT 27X-1 cells in different culture conditions.Transfected cells were seeded at a density of ~25,500 cells/cm² andcolonies established under puromycin selection for 10-15 days, in thepresence and absence of conditioned medium (+/−CM). Colonies were scoredfor a tight, medium or loose phenotype with respect to pluripotency,following Leishman's staining. Floxed hPDF colony scores shown are takenfrom electroporation transfection plates. Colony Floxed hPDF Floxed hPDFGFP control GFP control Morphology +CM −CM +CM CM Tight 49 58 23 20Medium 33 27 44 43 Loose 18 15 33 37 Total count 100 100 100 100

1-92. (canceled)
 93. A method of reprogramming or increasing the potencyof a mouse or human cell comprising specifically increasing expressionof a gene in the cell that encodes a factor which acts intracellularlyand maintains or confers pluripotency on a cell in the absence of gp130activation, wherein the factor is a polypeptide comprising the sequenceof SEQ ID NO: 4, thereby reprogramming or increasing the potency of thecell.
 94. The method of claim 93, wherein the gene has been introducedinto the cell.
 95. The method of claim 93, wherein the gene is locatedon an episomal plasmid or is stably integrated.
 96. The method of claim93, wherein the factor reprograms or increases the potency of a mouse orhuman cell.
 97. The method of claim 93, wherein the factor maintains orconfers pluripotency on a cell in the absence of LIF.
 98. The method ofclaim 93, wherein the factor maintains or confers pluripotency on a cellin the absence of feeder cells and in the absence of feeder cellextract.
 99. A vector comprising a nucleotide sequence encoding a factorwhich acts intracellularly and maintains or confers pluripotency on acell, wherein the factor is a polypeptide comprising the sequence of SEQID NO:
 4. 100. The vector of claim 99, wherein the factor maintains orconfers pluripotency on a cell in the absence of gp130 activation. 101.The vector of claim 100 comprising a promoter sequence.
 102. The vectorof claim 101 comprising sequences which enable removal or inactivationof the promoter.
 103. The vector of claim 99 for transfection of a cellsuch that the transfected cell may be maintained in a pluripotent state.104. The vector of claim 99 for transfection of a cell such that thetransfected cell is reprogrammed or the potency of the transfected cellis increased.
 105. A vector comprising a nucleotide sequence encoding afactor which acts intracellularly and promotes self renewal of apluripotent cell, wherein the factor is a polypeptide comprising thesequence of SEQ ID NO:
 4. 106. A vector comprising a nucleotide sequenceencoding a factor which acts intracellularly and inhibitsdifferentiation of a pluripotent cell, wherein the factor is apolypeptide comprising the sequence of SEQ ID NO:
 4. 107. A cellcontaining a vector comprising a nucleotide sequence encoding a factorwhich acts intracellularly and maintains or confers pluripotency in acell, wherein the factor is a polypeptide comprising the sequence of SEQID NO:
 4. 108. The cell of claim 107, wherein the factor maintains orconfers pluripotency in a cell in the absence of gp130 activation. 109.The cell of claim 107, wherein the vector is maintained as an episomalplasmid or is stably integrated.
 110. The cell of claim 107, whereinexpression of the factor results in the cell being maintained in apluripotent state.
 111. The cell of claim 107, wherein expression of thefactor results in the cell being reprogrammed or the potency of the cellbeing increased.
 112. A cell which is the progeny of the cell of claim107.